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COLORIMETRIC METHODS OF ANALYSIS Including Some Turbidimetrit Nephelometric Methods and By FOSTER DEE SNELL, P
COLORIMETRIC
METHODS
OF
ANALYSIS
Including Some Turbidimetrit
Nephelometric Methods
and
By
FOSTER DEE SNELL, P h.P ·
and
TB.1RD ED1Tl0N
VOLUME
IV
OMANIe-II
NBSS&LUP
\
Regional Centre Llbrary
Baogaio re 560 024 .
Acces~ioONo .1.~
D. VAN NOSTRAND COMPA~Y, INC.
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Copyright © 1954, by
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First Printing, September, 1954
Second (Prepublication) Printing, September, 1954
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,07 J_ ··· " Acces ioo ." • THE S. S. BOOK EMPORIU~ ·MOUNT-JOY" ROAO.
,07 J_ ··· " Acces ioo ." • THE S. S. BOOK EMPORIU~
,07
J_
···
"
Acces ioo
."
THE
S. S. BOOK
EMPORIU~

·MOUNT-JOY" ROAO. BASAVANGUO

BANGALORE-4

(INDIA)

PREFACE TO THE THIRD EDITION As in the second edition the aim of this edition
PREFACE
TO
THE
THIRD
EDITION
As in the second edition the aim of this edition has been completeness
but so many new methods appear in this field that much condensation
i necessary. We have attempted to include, or to refer to, tbe methods
that have been published since the second edition, up to the time of
publication. This has meant deleting many of the older references.
Alternative methods for a particular substance are still given, because
a method suitable for one purpose may not be suitable for another. For
example, some methods are for determination of 0.0001 mg. or less,
others for the order of 0.1 mg. of active ingredient. Some methods are
very precise, others much less so.
Substances that interfere with a determination are usually men-
tioned in the introductory paragraph. Available methods for removal
of these are described under" sample."
The preparation of samples i
given in great detail because much of the applicability of a method
depends on proper treatment of the sample to remove substances which
would interfere with the color development, and on preparation of a
solution with a suitable concentration of active ingredient.
Because so much detail must be given, explanatory matter is con-
densed as much as possible. It is expected that the reader will be abl e
to decide from th intJ'oductory paragraph, which explains the principle
and limitations of each method, which one to choose among alternative
methods.
This volume contains all remaining orO'anic and biological material
which could not be incorporated in the third volume because of space
limitations. Chapters in Volume III cover Hydrocarbons; Alcohol and.
their esters; Phenols; Quinones; Oxygen cycles, oxides, and peroxides;
Pentoses; Hexo es and Heptoses; Polysaccharides; Glucosides; Alde-
hydes; Ketones; Aliphatic acids and their e tel'; yclic acids and their
esters; omplex acids and derivatives; Sulphur derivatives; and IIalogen
compounds.
ACknowledgement is made of editorial as istance by Sally Cohen
and Elsie Testa, and of secretarial help by Dorothy Bevilacque Kruk
iii
lV PREF ACE TO THE THIRD EDITION and others. Acknowledgement to all who have assisted
lV PREF ACE TO THE THIRD EDITION
and
others.
Acknowledgement
to
all
who
have
assisted
would
be
impossible.
FOSTER DEE
NELL
CORNELIA T. SNELL
New York, N. Y.
August, 1954
CONTENTS CHAPTER 1 NITRITES, NITRATES, AND NITRO COMPOUNDS ••••••••••• 1 2 ALIPHATIC AMINES
CONTENTS
CHAPTER
1
NITRITES, NITRATES, AND NITRO COMPOUNDS
•••••••••••
1
2
ALIPHATIC AMINES AND AM IDES
••.••••••
•.•• .
29
3
AMINO ACIDS
••
•••••
••••••••.••••••
104
4
PROTEINS
170
5
AROMATIC PRIMARY,
SECONDARY, AND TERTIARY AMINES
197
6
Azo
COMPOUNDS,
lTROGEN-CONTAINING CYCLES,
238
7
UREA
AND
RELATED
COMPOUNDS
••.•
••••
.
.
317
8
COMPOUND
WlTH INOlWANIC RADICALS
•••••••••••••
343
9
STEROLS
361
10
HORMONES
•.
387
11
ALKALOIDS
427
12
ENZYMES
•.••••••
.•
••
487
13 ANTlliIOTJCS
535
14 AND RELATED
HEMOGLOBIN
COMPOUNDS
558
15 NATURAL
PIGMENTS.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
579
16 COLOR OF LIQUIDS
•••
•.
598
AUTHOR INDEX
.
.
.
.
.
.
• .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.•
605
SUBJECT
INDEX
••••••••••••••••••••••••••••••••.••••
637
TABLES PAGE TABLE 1. CHROMATOGRAPHIC SEPARATIONS • •••••• ••. ••• •••••. •
TABLES
PAGE
TABLE
1.
CHROMATOGRAPHIC
SEPARATIONS
••••••
••. •••
•••••.
13
2.
ABSORBANCE OF SOLUTIONS OF 2,4-DINITROPHENYL DERIVATIVES
OF AMINES
.
.
.
.
• •
• • •
• •
.
.
.
.
.
.
.
.
.
.
.
.
.
• • •
.
.
.
. •
30
3.
QUA NTITATIVE NATURE OF
THE
REACTION
BETWEEN
VARIOUS
AMINES AND SODIUM 1,2-NAPIITHOQUINONE-4-
ULFONATE.
32
4.
OPTIMAL CONDITIONS FOR CONVERSION OF AMlDES INTO HYDRO-
XAMIC ACIDS
3:1
5.
EXAMPLES OF MODIFIED PROCEDURES SUITABLE FOR COMPOUNDS
NAMED IN THE MEDIA LISTED
•.•
••••
,
6(;
6.
OPTICAL DEN S ITY DIFFERENCE FROM BLOOD EXTRACT
] 01
7.
DIFFERENTIATION
OF
SEVERAL
REPRE ENTATIVE
BARBITURATES
BY THEIR DIFFEREN CE RATIOS
.•
•.•••.•.•
102
8.
CHARACTERISTIC
DIFFEREN
ES OF BARBITURATES
••
••••••••
10 3
9.
DEGREE OF COLOR DEVELOPMEN T WITH NINHYDRIN
••.•
106
10.
WAVE LENGTH OF
COLOR REA CTION OF SPECIFIO PROTEINS WITH
11.
NINHYDRIN
ALANINE EQUIVALENCE OF
•••••
108
COPPER
SALTS
OF
DIPEPTlDES
AND
TRTPEPTIDES MEASURED AT 230 mil-
173
12.
ALANINE EQUIVALENCE OF COPPER SALTS OF 0.0003 M SOLU-
TIONS OF AMINO A CIDS MEASURED AT
230
mil,
.
.
.
.
.
.
.
.
.
183
13 .
WAVE LENGTHS FOR READING VARIOU S ANTIIIISTAMINES •
•. 281
14.
FACTORS TO GIVE l\ll CROMOLES OF ARGININE DECOM P OSED BY
SAMPLE AT 25 0
495
•••••••••••••••••• •••• •••••••••••••••
15.
F AOTORS FOR COLORIMETRIC TIMING METHOD .•.•.••
••.•••
533
16.
HEMOGLOBIN IN TERMS OF PERCENTAGE EQUIVALENTS AC CORD-
17.
18.
.•
CONVERSION FACTORS FOR MM. TO GRAM S HEMOGLOBIN PER
100 ml. OF WnOLE BLOOD USING NEWOOMER DISC
CORRECTION FACTORS FOR 'rIME OF STANDING IN NEWOOMER
ING TO VARIOUS STANDARDS
•.•.•••.
560
568
l\1ETHOD FOR II~OGLOBIN •
•••
••
••
569
vi
ILLUSTRATIONS NUMBER PAGE 1. Apparatus for extraction of fumigant • 4 2. Apparatus for distillation
ILLUSTRATIONS
NUMBER
PAGE
1.
Apparatus for extraction of fumigant
4
2.
Apparatus for distillation of nitroxylenol
9
3.
Apparatus for separation of dimethylamine and monomethyl-
amine
38
3.
Apparatus used fo1' both quantitative absorption of thiamine
by zeolite and sub. equent quantitative elution
87
5.
Sim1l1ified allllarattlS for sor1}tion and elution of thiamine
88
6.
Distillation unit for the concentration of urin
in vacuo
92
7.
Apparatus for aergtion of acetaldehyde from refluxing reac-
tion mixture
116
8.
Hydrogen filling apparatus
132
9.
Distillation apparatus
152
10.
Apparatus for oxidiltion of i oleucine to methylethyl ketone
160
11.
Apparatus for chromatography
25G
12.
Apparatus for delivery of sample
255
13.
Reduction-distillation apparatus for determination of maleic
hydrazide
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
264
14.
Exchange tube a semblies
304
15.
Extraction and separation of estrogens from androgens
391
16.
Separation of estrogens from phenols volatile with steam
403
17.
Separation of estriol from estrone-estradiol fraction
404
18.
Separation of estrone from estradiol
405
19.
Extraction apparatus
•.
•.•
474
20.
Distillation appara.tus
••.
549
21.
ahli hemometer
565
22.
Nomograph for correction of hemoglobinometer readings for
tim~ ?;.1'l~t~·ll';~~~'i.t'M~ •.•••••••••••••••••••••••••••••
~'\
CHAPTER 1 NITRITES, NITRATES, AND NITRO COMPOUNDS 1 THIS group of compounds is more heterogeneous
CHAPTER 1
NITRITES, NITRATES, AND NITRO COMPOUNDS 1
THIS group of compounds is more heterogeneous than may appear
superficially. The aliphatic nitrites and nitrates are really esters of
aliphatic alcohols. In contrast, the aromatic nitro compounds are very
stable and are a hasis for production of many other compounds.
Many of the nitro compounds give colors in alkaline solution, a very
simple method of development of color. Others react similarly in the
presence of metallic ions. Still others are read in ketone solutions. If
necessary, they are reduced to amines, diazotized, and coupled.
PRIMARY
ALIPHATIC
NITROPARAFFINS
If a solution of a primary nitroparaffin is made strongly alkaline, the
nitroparaffin is converted to the aci form. If then it is made strongly
acid with hydrochloric acid and ferric chloride added, the r ed color is
suitable for estimation 2 of amounts as low as 0.5 mg. An optimum final
pH is 1.25-1.3; if more acid, the color fade gradually; if less acid, the
color is brownish red. Thus the amounts of reagents specified in the
procedure should be checked when a batch is prepared to insure the
optimum pH. The reaction has been effectively applied to nitroethane,
I-nitropropane, and I-nitrobutane. Nitromethane does not react; the
color with 2-nitropropane and 2-nitrobutane fades rapidly. Most
phenols, enols, and many aliphatic acids interfere. A red color of
tetranitromethane with highly un aturated fatty acids in chloroform or
carbon tetrachloride solution has not been developed to quantitative
application. s Nitromethane gives color with vanillin in ammoniacal
solution.
The alkali salts of the primary aliphatic nitro compounds couple with
diazonium salts to produce azo compounds, which change to colored
1 See Chapter 1, Volume III, for details of organization, condensation, etc.
2 Eugene W. Scott and Joseph F. Treon, Ind. Eng. C1trm., Anal. Ed. 12,
189·90 (1940).
8 H. P. Kaufmann, Bao Wei King, and Lan·Sun Huang, Ber. 75B, 1201·14 (1942) .
1
2 NITRITES, NITRATES, AND NITRO COMPOUNDS phenylhydrazones. 4o The introduction of a sulfo group is
2 NITRITES, NITRATES, AND NITRO COMPOUNDS
phenylhydrazones. 4o The introduction of a sulfo group is needed to make
the dye sufficiently water-soluble. Diazotized sulfanilic acid is a suitable
reagent.
Since an equilibrium exists between the azo compound and the hydra-
zone, the color is dep ndent on pH. By addition of a buffer, pH is
maintained at 6 and stable colors obtained. Nitrornethane then gives a
cherry-red color and nitrocthane and I-nitropropane yellow. Thus it is
possible to determine nitromethane in the presence of other nitropar-
affins. olors are constant for about 15 minutes; thereafter, nitroethane
deepens in color and nitromethane and 1-nitropropane fade. Secondary
nitroparaffins and alkylnitriles do not interfere. The reaction will detect
0.001 mg. of nitromethane or 0.005 mg. of 1-nitropropane pel' mI. The
color conforms to Beer's law over the range of 0.01-0.1 per cent. Accur-
acy is in the range of ±1-4 per cent.
Sample-Gas. Shake an appropriate size of sample with 2 per cent
sodium hydroxide olution and u e all or an aliquot of the resulting
solution for development with ferric ion.
Procedure-By fet'ric ion. Measure out an aliquot of sample con-
taining 1-20 mg. of nitroparaffin, neutralize, and dilute to about 15 ml.
Add 1.5 ml. of 20 per cent sodium hydroxide solution and let tand for
15 minutes. Add 6 mi. of 1: 7 hydrochloric acid and at once 0.5 mI. of
10 per cent ferric chloride hexahydrate. Dilute to 25 mi. and read at
500 mp, against a reagent blank. If the blank is too large relative to
the test substance, reduce the amount of ferric chloride added. Read
against a calibration curve prepared with the same compound.
By diazotized sulfanilic acid. Make up a buffer solution of 15 grams
of glycocoll and 11.70 grams of sodium chloride per liter of water
containing 50 mI. of 1.12 per cent potassium hydroxide
solution.
.As
diazonium reagent mix equal volumes of 0.9 per cent sodium nitrite
solution and 0.72 per cent sulfanilic acid solution containing 1.8 ml. of
concentrated hydrochloric acid per 100 mi. Mix 1 ml. of nitroparaffin
solution in methanol with 2 ml. of buffer solution. With agitation add
5 mI. of the diazonium reagent and read immediately at an appropriate
wave length according to the color developed.
4 F. 'l'urba, R. Haul, Ilnd G. Uhlen, Llngcw. Chern. 61, 74·5 (1949).
SECONDARY ALIPHATIC NITROPARAFFINS 3 PRIMARY ALIPHATIO DINITROPAR.AFFINS At pH 7 the alkali salts of
SECONDARY ALIPHATIC NITROPARAFFINS
3
PRIMARY
ALIPHATIO
DINITROPAR.AFFINS
At pH 7 the alkali salts of 1,1-dinitropropane are readily formed
and are an intense canary-yellow.1> At this pH any salts of primary
mononitroparaffins are colorless.
Procedure-Add 2 ml. of 4 per cent sodium hydroxide solution to
3 ml. of a 0.003-0.2 per cent solution of 1,1-dinitropropane in 50 per
cent methanoL Read at once with a filter centering around 430 mil.
SECONDARY
ALIPHATIO
NITROPARAFFINS
The difference in reaction of a primary nitroparaffin and a secondary
nitroparaffin, ~CHN02' is sufficient to give a distinctly different re-
action. The latter r eact with nitrous acid to produce p eudonitrols which
are colorless and insoluble in both acid and alkali. Dissolved in toluene,
they give a blue color.G The method detects 0.25-1.25 per cent by weight
with not over ±6 per cent error. The color begins to fade after 15
minutes. Primary nitroparaffins and alkylnitrites do not interfere.
Most secondary and some tertiary nitroparaffins react with resorcinol
in the presence of concentrated sulfuric acid to give an intense red-blue
color. 7 Primary nitroparaffins uniformly are negative. The reaction is
decomposition of the nitroparaffin by the concentrated ulfuric acid to
form successively nitrous acid and nitrosyl sulfuric acid. By reaction
with the resorcinol this forms a p-nitro ophenolic compound. Phenol,
orcinol, pyrogallol, pyrocatechol, and N- (I-naphthyl) -ethylenediamine
dihydrochloride give the reaction. Oxidizing and reducing agents must
be absent. The method will detect 0.001 mg. and determine as l ow
3-5 times the amount.
The reaction is given by 2-nitropropane, 2-nitro-2-methylpropane, 2-
nitrobutane, l-chloro-l-nitrobutane, trichloronitromethane, l-chloro-1-
nitroethane, 1,l-dichloro-l-nitroethane, l-chloro-1-nitropropane, 2-chloro-
2-nitropropane, 1,1-dichloro-1-nitropropane, monobromonitromethane,
I-bromo-l-nitroethane, I -bromo-l-nitropropane, 1-bromo-nitrobutane,
2-bromo -2-nitropropane, 2- bromo -2-nitrobutane, 1,1-dibromo-1-nitro-
ethane, l,l-dibromo-l-nitropropane, 1,1-dibromo-1-nitI'obutane, 2-nitro-
as
r; ibid.
6 Ibid.
7 Lawrence R. Jones and John A. Riddick, Anal. Chem. 24, 1533· 6 (1952).
4 NITRITES, NITRATES, AND NITRO COMPOUNDS 2-chloro-l,1-bis (p-chloropbenyl) propane, 2-nitro-l,1-bis (p-chlorophenyl)
4 NITRITES, NITRATES, AND NITRO COMPOUNDS
2-chloro-l,1-bis (p-chloropbenyl) propane, 2-nitro-l,1-bis (p-chlorophenyl)
propane, and 2-nitro-l,1-bis(p-chlorophenyl) butane.
There is no reaction by nitromethane, nitroethane, 1-nitropropane, 1-
nitrobutane, 2,2-dinitropropane, I-nitro-2-chloroethane, 2-nitro-2-bromo-l-
methoxy-1-phenylpropane , p-nitrostyrene, 1- (p-nitrophenyl) -2-nitropro-
pene, 1- (p-cblorophenyl) -2-nitro-1-propanol, 1- (p--cblorophenyl-2-nitro-
1-butanol, trihydroxymethylnitromethane and 2-nitro-2-bromo-l-phenyl-
ethylene.
-
Air
Fro. 1. Apparatus for extraction of fUU1i~ant
A. Aeration
tube.
Inside tube.
67 <'ID. long,
o.d. 32
mm .• i.d. 28mm .• wound with
43 turns of No 22 Nichrome V resistance wire lh inch between turns Outside jacket.
57 em. long, o.d. 45 mm. B. Air scrubbing bottle. tilled with sulfuric acid. sp. gr.
1.84. C. Standard taper 24/40 glass joints. D. Leads to Variac. E. Tygon tubing.
F. Flowmeter, Fisher Laboratory ModeJ No. 11-163. G. Glass wool plug_ T. TraPB.
25 x 200 mm., borosilieato tubes, containing 10 ml. of sulfuric Acid. sp. gr. l.84.
SampJe-2-Nitropropane in air.
Pass tbe air at 1 liter per minute
through 3 successive absorbers (tubes T I , T 2 , and Ts of Fig. 1) each
containng 10 mL of concentrated sulfuric acid.
Recovery is quantita-
tive.
After an
appropriate amount of air has been passed,
combine
the contents of the 3 absorbers and take an aliquot for development
with resorcmol.
NitroparajJin mixtures.
Dissolve a weighed sample in concentrated
sulfuric acid for development of an aliq-uot with resorcinol.
1,I-DICHLORO-I-NITROETHANE, ETHIDE 5 Solutions of nitroparajfins. Place an appropriate portion of the solution in A
1,I-DICHLORO-I-NITROETHANE, ETHIDE
5
Solutions of nitroparajfins. Place an appropriate portion of the
solution in A of Fig. 1 and pass a current of air until the nitroparaffin
is transferred to the absorption tubes. Combine the contents of the
3 absorbers and take an aliquot.
Procedure-By nitrous acid. Add 5 ml. of a fresh 10 per cent
solution of sodium nitrite to 10 ml. of a solution which contains 0.025-
0.125 gram of the secondary aliphatic nitropropane in 40 per cent
methanol, and cool in ice for 3 minutes. Mix with 15 ml. of toluene
and add 2 ml. of 1: 6 sulfuric acid drop wise with shaJcing. When the
aqueous layer is decolorized, separate the toluene laye r. Filter
through
a dry filter and read around 610 mf.-t within 10 minutes.
By res01·cinol. Dilute an aliquot of sample in concentrated sulfuric
acid to contain about 0.02 mg. of test substance in 10 ml. Stopper
the tube and heat in boiling water for 5 minutes. Cool at 20-25° for
2-3 minutes and stratify on the surface 5 ml. of 1 per cent aqueous
resorcinol on top. Mix carefully, restopper, and heat in boiling water
for 10 minutes. Cool and read at 560 mf.-t against a reagent blank.
1 ,I-DIOHLORO-l-NITROETHANE, ETHIDE
Insofar as the nitro group is concerned, ethide acts as a tertiary
nitro compound. It responds to the method for secondary aliphatic
nitro compounds by reaction with resorcinol 8 preceding, and is so
det ermined on samples of 0.005-0.1 mg.
Sample-Corn. Transfer a sample of 50-500 grams to tube A of
Figure 1. It will expedite the vaporization to heat tube A to 50-55°.
Put 10 ml. of concentrated sulfuric acid in each of the absorbers and
pass air at 1 liter per minute for 15 minutes. Replace the first absorp-
tion tube with another containing 10 ml. of concentrated sulfuric acid
and
r epeat. Continue thus until all of the test substance has been
vaporized and absorbed as determined by development of each tube
as removed from the apparatus. Finally, develop an aliquot of the
contents of each of th e three absorbers used in the final stage of
volatilization of the sample.
Procedure-Follow that described for the secondary nitroparaffins
by resorcinol above.
8 Ibid.
6 NITRITES, NITRATES, AND NITRO COMPOUNDS AMYL NITRITE A characteristic red color of amyl nitrite
6 NITRITES, NITRATES, AND NITRO COMPOUNDS
AMYL NITRITE
A characteristic red color of amyl nitrite with phenol, copper
sulfate, and acetic acid permits its estimation.o Results are, in general,
accurate to ±
10 per cent.
Procedure-Premix a reagent containing 24 mI. of 5 per cent aqueous
phenol, 1 ml of 2 per cent copper sulfate pentahydrate solution, and
5 ml. of glacial acetic acid.
Procedure-Gases. Measure 30 ml. of reagent into an absorber
having a sintered-glass "}:>late with an average opening size of 5 microns.
Pass through a known appropriate volume of gas at 40 ml. per minute
until a readable color can be obtained. Heat at 80° for 5 minutes and
read at 520 mp. against a reagent blank.
Alternatively add 6 ml. of reagent to a gas buret containing about
300 ml. of sample. Shake mechanically for 15 minutes and withdraw
the reagent. Heat as before and read.
AMYL NITRATE
Nitric acid esters as well as nitrates are estimated by fixation of
the nitric acid as a nitroxylenoPo The color is developed in alkaline
solution. Previously the reaction has been developed with 2,4-xylenol
(Vol. IT, p. 794) but 3,4-xylenol is preferabl e. Stabilizers such as
diphenylamine, diethyldiphenylurea, or their derivatives may be present.
The end product is 6-nitro-3,4-xylenol. Nitrites mu st be absent. The
method has also been applied successfully to decyl nitrate, and would
doubtless be applicable to others. Hydrolysis of nitrocellulose is
incomplete.
Procedure-Dissolve a sample containing 0.07-0.1 gram of n-amyl
nitrate in petroleum ether boiling at 30- 60° and dilute to 100 ml. Mix
a 5-ml. aliquot with 1 ml. of 2 per cent solution of 3,4-xylenol in
acetone and add 15 ml. of sulfuric acid prepared by mixing 66 ml. of
concentrated acid with 30 mI. of water. Shake mechanically with the
stopper fastened down, for 30 minutes. Cool in ice and dilute by addition
to 80 ml. of water. Rinse the flask with water and acetone. Steam-
9 R. G. Horswell and Leslie Silverman, Ind. Eng. Chcm.,
Iinal.
Eel.
13, 555-8
(1941).
10 Albert O. Holler and Rita V. Buell,
Iinal.
Cham. 21, 1385· 9
(1949).
ETIIYLENE GLYCOL DINITRATE 7 distil the product from an oil bath and collect the distillate
ETIIYLENE GLYCOL DINITRATE
7
distil the product from an oil bath and collect the distillate in 5 ml.
of 2 per cent sodium hydroxide. After distillation of about 75 ml.
cool and dilute to 100 ml. Filter through cotton on paper and read
at 420 m,.,. against a reagent blank.
DECYL NITRATE
A modiiication of the preceding method for amyl nitrate with
3,4-xylenol is applicable to decyl nitrate. A solution in petroleum
ether cannot be used because the large nonpolar group prevents com-
plete extraction by sulfuric acid.
Procedure-Add a sample containing 0.07-0.125 gram of decyl
nitrate to 5 ml. of 8 per cent solution of 3,4-A"Ylenol in acetone. Add
150 ml. of sulfuric acid prepared by mixing 66 ml. of concentrated
acid with 30 m}. of water. Shake mechanically with the stopper
fastened down for 30 minutes. Cool and dilute with the sulfuric acid
to 250 ml. If foaming tends to occur, add a drop or two of petroleum
ether. Mix well and add 10 ml. to 150 ml. of water. Complete as for
amyl nitrate starting at "Rinse the flask with water and acetone."
ETHYLENE
GLYCOL
DINITRATE
Ethylene glycol dinitrate is estimated by the usual nitrate color
with pbenoldisulfonic acid.u Color development is incomplete below
0.5 mg. of test substance in the sample; above 4.2 mg. the color is too
intense. The average error in that range is less than ±1 per cent.
Sample-Air. Pass a known volume of air containing ethylene
glycol dinitrate through two D-tubes cooled with acetone and solid
carbon dioxide. The ethylene glycol dinitrate condenses almost entirely
in the first tube. Carefully rinse the tubes with acetone and dilute
the washings to a known volume.
Procedure-As a rea"'ent dissolve 25 ml. of colorless phenol in 150
ml. of concentrated sulfuric acid. Trace of nitric acid present in
sulfuric acid are removed by agitating with mercury. Add 75 ml.
of fuming sulfuric acid containing 13 per cent of free sulfur trioxide.
Stir well and heat in a fla k in boiling water for 2 hours. The reagent
so prepared is free from mono- and trisulfonic acids.
11 John H. Foulger, J. Ind. Hyg. ToxiooZ. 18, 121·9 (1936).
8 NITRITES, NITRATES, AND NITRO COMPOUNDS Transfer a sample in acetone, containing 0.5-4.2 mg., to
8 NITRITES, NITRATES, AND NITRO COMPOUNDS
Transfer a sample in acetone, containing 0.5-4.2 mg., to a porcelain
dish and let the acetone evaporate spontaneously. Add 2 ml. of the
phenoldisulfonic acid reagent and stir with a glass rod. After 10
minutes dilute with water to about 75 ml. Add 1: 1 ammonium hydroxide
until alkaline, cool to room temperature, and dilute to 100 m!. Read
against a reagent blank at 490 m",.
GLYCERYL NITRATES
The method for nitrates by phenoldisulfonic acid is applied to
analysis of gIyceryl nitrates.12 They may also be distilled, reduced to
ammonia, isolated, and estimated by Nessler's reagent. 1S
Another technic is to hydrolyze glyceryl nitrates in 62.5 per cent
sulfuric acid and use the liberated acid for nitration of xylene. a This
is volatile with steam and read as an intensely yellow solution. The
pH must be above 11.1~ Interference occurs by large amounts of halogen
salts, metals reactive with the acid, nitrites, sulfides, and hydrogen
peroxide. Erythritol or pentaerythritol tetranitrates give the same
reaction.
Samples-Air. 16 Absorb that in a 5-liter flask by shaking with
50 ml. of ethanol. Remove the solution and heat to boiling to drive
off interfering impurities such as ethyl nitrite. Add 1 ml. of 17 per
cent potassium hydroxide solution and evaporate to dryness on a
water bath. From each mol of nitroglycerine, two mols of potassium
nitrite and one of potassium nitrate result. Take up in water, for
the use of aliquots. Develop with phenoldisulfonic acid or apply other
methods for nitrate and nitrite (Vol. II, pages 785-807).
TcibZets. Transfer crushed tablets equivalent to 1 mg. of glycerol
trinitrate to a stoppered cylinder. Usually 2 tablets are sufficient.
Add 5 ml. of glacial acetic acid and shake mechanically for an hour.
Filter and use an aliquot of the filtrate for development by phenol-
disulfonic acid.
Alternatively place 5 tablets in a 500-ml. Kjeldahl flask with
25 ml. of saturated sodium sulfate solution. Add 75 ml. of water and
12
H. O. Meek, Quart. J. Pharm. Pharmacol. 8, 375·7 (1935).
18
Wilfred Smith, Ibid. 8, 370·4 (1935).
14
Herman Yagoda, Inil. Eng. Chem .,
d.nal.
Ed.
15, 27·9
(1943).
15
Per Lundgren and Teodor Oanbiick, S1J(J'fI,sk Farm. Tid. 52, 298·304 (1948).
16
v. G. Gurevich, L. E. Karlson, and K. K. Sigalovskaya, Zavoaskaya Lab. 12,
553-4 (1946).
GLYCERYL NITRATES 9 sufficient 1:360 sulfuric acid to render acid to litmus paper, usually 0.3
GLYCERYL NITRATES
9
sufficient 1:360 sulfuric acid to render acid to litmus paper, usually
0.3 mI. Distil through a stillhead into 10 ml. of 0.4 per cent sodium
hydroxide solution. Continue until the distilling flask goes just to
dryness. Rinse the condenser and outlet tube with water and evaporate
the alkaline distillate to dryness. Add 2 ml. of water, 0.3 gram of
reduced iron, and 2 mi. of 1: 1 sulfuric acid. Let this react for 10
minutes, then boil for 2 minutes.
Transfer to steam distillation equip-
ment and add 4 ml. of saturated sod-
ium hydroxide solution. Distil the
ammonia into 10 ml. of 1: 360 sul-
furic acid. Collect nearly 500 ml. of
distillate and dilute to that volume
for development of aliquots with
Nessler's reagent.
Biological. Extract the sample
with ether and wash the ether extract
with 10 per cent sodium sulfate solu-
tion until the chloride reaction with
silver nitrate is negative. Dilute the
extract to a known volume for ali-
quoting and development as nitro-
xylenol.
Procedure-By phenoldisulfonic
acid. Transfer an aliquot of sample
in glacial acetic acid to a small por-
cerain
Add
at once about 2 ml.
of phenoldisulfonic acid reagent
(Vol II, page 792). Stir and let stand
FIG. 2.
Apparatus for distillation of
nitroxylenol
for 15 minutes. Dilute with about
8
ml.
of
water
and
add
1:3
ammonium hydroxide cautiously until just alkaline. Dilute to 20 ml.
at 20 0 and filter. Read at 490 mp. against a reagent blank.
By Nessler's reagent.
Use 100 ml. of ammonia distillate as sample.
Add 2 ml. of Nessler's reagent (page 181), and read at 420 mIL.
As nitroxylene. Select an aliquot of an ether solution of the sample
to contain not over 3 mg. of test substance. Evaporate the bulk of the
ether in a stream of cold air to avoid volatilizing the ester. Dissolve
the residue in 1 mI. of 2 per cent m-xylene in acetone. Add 15 ml.
10 NITRITES, NITR.ATES, AND NITRO COMPOUNDS of 5: 3 sulfuric acid and allow 30 minutes
10 NITRITES, NITR.ATES, AND NITRO COMPOUNDS
of 5: 3 sulfuric acid and allow 30 minutes for reaction at room tempera-
ture. Dilute with 100 ml. of water and steam distil into a water-cooled
Nessler tube as shown in Figure 2. Collect the distillate in 5 mI. of
2 per cent sodium hydroxide in the tube. Dilute to 50 mI. and read
against a reagent blank.
ERYTHRITOL
TETRANITRATE
This is determined by the procedure given for glyceryl nitrates as
nitroxylene.
PENTAERYTHRITOL TETRANITRATE
This is determined by the
procedure
given for
glyceryl
nitrates
with phenoldisulfonic acid 17 or as nitroxylene (page 9).
Sample-Waste liquors. If in an organic solvent, evaporate to
dryness and take up in chloroform. For aqueous solutions extract a
50-ml. sample with 10, 10, and 5 ml. of chloroform. Wash the com-
bined extracts with 5 mI. and 5 mI. of water. Evaporate the chloro-
form solution to dryness. Take up the residue in 2 mI. of glacial
acetic acid and complete as for glycerol nitrates (page 9) .
NITROGUANIDINES
Total nitroguanidines are read in the ultraviolet at 265 m,u.18
If
nitroaminoguanidine is also present, correct the value obtained for an
equivalent of this compound separately determined.
NITROAMINOGUANIDINE
The reaction of sodium or potassium hydroxide with an alcoholic
solution of nitrophenylhydrazone has been used to dctermine aldehydes
and ketones. 10 It is applied to alkaline solutions of phenylhydrazones
of phenylaromatic
aldehydes. 20
This is applicable to the estimation of
hydrazino groups in nitroaminoguanidine, aminoguanidine, diamino-
guanidine, and semicarbizide. This is applied to mixtures of nitro-
17 J. Allert, DaM'k Tid8. Farm. 22, 188·97
(1948); M. T. Golubeva,
Gigiena i
Sanit. 1952,
No.
.5, 47·9.
18 John E. DeVries and E. St. Clair Gantz, Anal. Ch em. 25, 1020·2 (1953).
19 G. R. Lappin and L. C. Clark, Ibid. 23, 541 (1951).
20 F. Cha.ttawa,., S. Ireland., a.nd A. Walker, J. (]hem. 800. 127, 1851·5 (1925).
NITROBENZENE 11 aminoguanidine with nitroguanidine. 21 Practically it is estimated as the hydrazones with
NITROBENZENE
11
aminoguanidine with nitroguanidine. 21 Practically it is estimated as
the hydrazones with p-nitl'obenzaldehyde at 385 m!1- where the absorption
by the excess aldehyde approaches zero. Nitroaminoguanidine and
nitroguanidine give maximum absorption at 267 mit and 265 m!1-.
ml.
Sample-Use an aqueous solution containing about 0.5-1.0 mg. per
They may be prepared by extraction with water on a steam bath
for 30 minutes at
90 0 •
Procedure-To 1 ml. of sample add 50 ml. of water, 5 drops of
concentrated hydrochloric acid, and 1 ml. of 2 per cent solution of
p-nitrobenzaldehyde in methanol. Heat to 70-80 0 for
3-5 minutes.
Cool and add 1 m!. of 56 per cent aqueous solution of potassium
hydroxide. Dilute to 100 ml. with water. After 15 minutes read at
385 m!1- against a reagent blank. No change in color intensity occurs
for days.
NITROBENZENE
By absorbing nitrobenzene in a suitable nitrating mixture it is
converted to m-dinitrobenzene, which is estimated in dry acetone solution
by the violet color developed with sodium hydroxide solution. 22 The
color development is slow unless the acetone is maintained nearly an-
hydrous. With more than 10 per cent of water, precipitation occurs.
Accuracy is to ±5 per cent. By increase of sodium hydroxide solution
the sensitivity is increased. Nitrobenzene dissolved in concentrated
sulfuric acid also gives a color suitable for estimation in the absence of
mononitrated toluenes. 2s
Nitrobenzene is also determined by reduction with stannous chloride
and application of methods for aniline (pages 197 to 919).
Sample--.A.ir.
Add 15 ml. of a 20 per cent solution of ammonium
nitrate in concentrated sulfuric acid to each of two absorption bottles.
Pass the gas through these in series at the rate of about 10 liters per
hour. Keep the tubes cooled in ice and pass not less than 40 liters of air.
21 John E. DeVries and E. St. Clair Gantz, Ibid. 25,1020·2 (1953).
22 A. S. Zhitkova., S. I. K!lplun, and Joseph B. Ficklen, "Estimation of
Poisonous Gases and Va.pors in the Air," pp. 173 ·5. Service to Industry, West
Hartford, Conn. (1936); M. S. Bykhovskaya, Org. Cllem. Ind. (USSR) 6, 638 ·9
(1939); I. M. Korenman and A. M. Fisher, Zavodikaya Lab. 14,1058· 60 (1948).
28 G. Halfter, Z. anat, Chem. 128,140-1 (1948).
] 2 NITRITES, NITRATES, AND NITRO COMPOUNDS POUT the nitration mixture into about 50 ml.
] 2
NITRITES, NITRATES, AND NITRO COMPOUNDS
POUT the nitration mixture into about 50 ml. of chilled distilled
water. Extract with two successive 10-ml. portions of benzene. Add
a saturated solution of sodium bicarbonate to the combined extracts
until neutralization is complete. Separate the benzene layer and dry
with anhydrous calcium chloride. Remove from the desiccating agent
and evaporate the solvent at room temperature on a watch glass. For
development with sodium hydroxide take up in anhydrous acetone for
the use of an aliquot. Such an air sample will, at the same time, convert
any azobenzene to p,p'-dinitrodiphenyldiimide which does not interfere.
Procedure-By sodium hydroxide in acetone. Measure an aliquot
in anhydrous acetone containing 0.05-0.2 mg. of nitrobenzene as dinitro-
benzene. Dilute nearly to 10 ml. with anhydTous acetone and add 0.2 ml.
of 40 per cent sodium hydroxide solution. Dilute to vol ume with
acetone, mix well, and after 5 minutes read at 570 mp against a reagent
blank. A suitable standard for development of a calibration curve
contains 0.01365 gram of recrystallized m-dinitrobenzene per liter of
acetone and is equivalent to 0.01 mg. of nitrobenzene per ml.
NITRO AND NITROSO
PHENOLS AND ANALOGOUS
COMPOUNDS
The nitro and nitroso compounds are separated chromatographically
and read in the ultraviolet.24 As developed it is applicable to five
pairs of C-nitro and C-nitroso compounds shown in Table 1. Application
to N-nitroso compounds is probable. Specific methods for 0 - and
p-nitrophenol are in Vol III, pages 124-125.
1-PaOPYLOXY-2-AMINO-4-NITROBENZENE
This sweetening agent is developed by r eduction to the amine with
iron, diazotizing, and coupling with a-naphtho}.25
Procedure.-Tablets. Dissolve a tablet in 100 ml. of 1: 120 hydro-
chloric acid. Add 0.1 gram of iron filings, filtering after reaction is
complete. Add 1 ml. of freshly prepared 0.01 per cent sodium nitrite
solution, and after 10 minutes add 1 ml. of 0.35 per cent solution of
a-naphthol in 0.4 per cent sodium hydroxide. Read against a reagent
blank.
24 D. N. Gullstrom, H. P. Burchfield and J. N. Judy, Ind. Eng. Chem., .4nal.
Bd.
18, 613·6
(1946) j
W. R. Edwards, Jr. and Oilton W.
Tate, .4Ml.
Chem.
23,
826·30 (1951).
21} Klement Mohler, Z. Lebena1ll
17nters'Uch. 1£. Forsch. 91, 124·6 (1950).
2,3,5,6-TETRACHLORONITROBENZENE 13 TABLE 1. CHROMATOGRAPIDO SEPARATIONS Compound Solvent Sorbent Developer
2,3,5,6-TETRACHLORONITROBENZENE
13
TABLE 1.
CHROMATOGRAPIDO SEPARATIONS
Compound
Solvent
Sorbent
Developer
p-Nitrophenol
Benzene
2: 1 Silicic acid
and Oelite
2 :1 Silicic acid
and Celite
2 :1 Silicic acid
and CeJite
2: 1 Silicic acid
and Celite
Silicic acid
Silicic acid
2 :1 Silicic acid
and Celite
96:4 Benzene-
acetone
p-Nitrosophenol
Benzene
96:4 Benzene-
acetone
2,4-Dinitroresorcinol
Benzene
96:4 Benzene-
acetone
2,4-Dinitrosoresorcinol
Benzene
96:4 Benzene-
acetone
1-Nitro-2-naphthol
Benzene
Benzene
1-Nitroso-2-na.phthol
Benzene
Benzene
2-Nitro-1-naphthol
Benzene
Benzene
2-NitrOBo-1-naphthol
Benzene
2 :1 Silicic acid
Qnd Celite
Benzene
2,2-Dinitropropane
Petroleum
ether
Silicic
acid
2-Nitroso-2-nitropropane
Petroleum
ether
Silicic
acid
Diethylnitrosoamine
Petroleum ether
Silicic acid
1 :1 Benzene-
petroleum-ether
1 :1 Benzene-
petroleum-ether
Petroleum ether
2,3,5,6-TETRACRLORONITROBENZENE
The chlorine atom ortho to the nitro group of 2,3,5,6,-Tetrachloro-
nitrobenzene is labile, permitting formation of the intense color of a
nitroquinoid ion_20 The color develops with tetraethylammonium hy-
droxide in about 8 minutes and starts to fade after about 20 minutes.
The purplish red has a peak at about 548 mp
Anhydrous conditions
are necessary. Benzene may replace part of the acetone used as solvent.
The tetrachloronitrobenzene also reacts with a-naphthylamine and
procaine hydrochloride to give an appropriate color for reading. 27
Procedure-By
tetraethylammonium hydroxide.
Dilute a sample
containing up to 0_05 mg. of test substance in acetone to 10 ml. with
acetone. Mix and add 0_1 ml. of 2 per cent freshly diluted tetraethyl-
ammonium hydroxide and mix. After 10 minutes but before 20 minutes,
read at 550 mp. against a reagent blank.
20 M. E. Auerbach, .dnal. ChC'lIi. 22, 1287-8 (1950).
27 Teodor Canback and Halina Zajaczkowska, J. Pharm. Pharmacol. 2, 545-8
(1950).
14 NITRITES, NITHATES, AND NITRO COMPOUNDS By a-naphthyLamine. As a reagent, dissolve 0.2 gram of
14 NITRITES, NITHATES, AND NITRO COMPOUNDS
By a-naphthyLamine. As a reagent, dissolve 0.2 gram of a-naphthyl-
amine in 30 ml. of boiling water and filter. Wash the filter twice with
50-ml. portions of hot water, add 50 ml. of glacial acetic acid, and
dilute to 150 ml.
Reflux a sample containing 0.05-0.3 mg. of 2,3,5,6-tetrachloronitro-
benzene in 5 ml. of acetone with 5 ml of 2.8 per cent ethanolic potassium
hydroxide for 15 minutes. Cool and add 5 ml. of 3 per cent procaine
hydrochloride in 15 per cent acetic acid. Add 5 ml. of the a-naphthyl-
amine reagent and mix. After 30 minutes dilute to 25 ml. with ethanol
and read at 500 mp. against a reagent blank.
NITROTOLUENES
Nitrotoluenes give color when dissol ved in concentrated sulfuric
acid. The ortho- and meta- isomers give the same yellow color and the
para- compound gives yellowish-green. 28 The color intensity is increased
threefold by a change of acid concentration from 97 per cent to 100
per cent. Protected from moisture absorption and light, the color is
stable for 48 hours. The natural yellow color of the compounds prac-
tically offers no interference and more highly nitrated toluenes give
colorless solutions. Nitrobenzene interferes.
The blue color of 2,4-dinitrotoluene with alcoholic sodium hydroxide
in the presence of m-dinitrobenzene is used to estimate less than 0.3
per cent of nitrotoluene in nitrobenzene. 2o 2,6-Dinitrotoluene gives a
red color under the same conditions. A red color from dinitrothiophene
disappears with excess of alcoholic sodium hydroxide solution.
Procedure-By sulfuric acid. Dissolve a sample containing 1 to 30
mg. of these nitrotoluenes in concentrated sulfuric acid and dilute to
100 ml. Read at 436 mp, and compare with a curve prepared with
the same batch of sulfuric acid. Roughly m-nitrotoluene gives half the
absorption of the 0- and p-isomers. Read the foregoing solution at
305 mp, as the p-isomer and subtract the value to get the m-isomer.
2,4,-dinitrotoluene. Mix 5 ml. of a sample of nitrobenzene con-
taining nitrotoluene with 20 ml. of concentrated sulfuric acid. Add
slowly, at a temperature of 40°, 32 ml. of a mixture containiug 20 ml.
of concentrated sulfuric acid and 12 ml. of concentrated nitric acid.
28 G. HaUter, Z. anal. Cloem. 128,140-1 (1948).
29 H. Muraour, Bun. BOC. chim. 43, 71-S (1928).
/3-NITRONAPHTHALENE 15 After a few minutes pour into water to precipitate m-dinitrobenzene and dinitrotoluene. Extract
/3-NITRONAPHTHALENE
15
After a few minutes pour into water to precipitate m-dinitrobenzene
and dinitrotoluene. Extract with ether and wash the ether extract
with water. Then wash with 2 per cent sodium hydroxide solution,
and again with water. Dilute the ethereal solution to 200 ml. with
ethanol. Treat 10 ml. of this with 10 ml. of a saturated alcoholic
solution of sodium hydroxide. Read the color against a reagent blank.
a-NITRONAPRTRALENE
When dissolved in concentrated sulfuric acid, a-nitronaphthalene
gives a blood red color suitable for its estimation in the presence of the
p-isomer and more highly nitrated naphthalenes. so The concentration
of acid is important. The colors given by nitrotoluenes can be screened
out.
fi-NITRONAPRTRALElNE
By sulfonation, reduction, and measurement of fluorescence, fi- nitro-
naphthalene is measured in a-nitronaphthalene, whether crude or
refined. S1 Both treatments are necessary to separate the a- and fi-isomers.
Procedure-Nitronaphthalenes. Mix 0.1 gram of sample with 1 ml.
of 99-100 per cent sulfuric acid. Dissolve for about 30 seconds at 50°,
cool to 25 0, and add 2 ml. of 58-60 per cent fuming sulfuric acid,
with vigorous agitation. Digest at 100° for 5 minutes, chill in ice, and
cautiously add 2-3 ml. of water. Add to 25 ml. of water and dilute
with rinsings to 50 ml. Add 0.5 gram of activated carbon, boil for 5
minutes, and filter while hot through a Buchner funnel. Cool and dilute
to 250 ml. The color is yellow to brownish. Decolorize with 0.5 gram
of Filter-eel and filter by gravity.
Heat 20 ml. with 0.5 gram of zinc dust on a steam bath for 5 minutes.
Cool, dilute to about 80 ml., and add 4 ml. of 50 per cent sodium acetate
trihydrate solution. Dilute to 100 ml. and filter. The color should be
no more than light yellow, the pH about 4.6. Read the fluorescence
with a Coleman Universal spectrophotometer, using a UV-1 light
filter for the mercury lamp and filters 5433 and 3850 for the photo-
electric cell.
so G. Haliter, Z. anal. Ghem. 128, 140·1 (1948).
31 F. L . English and J. W. Eppert, .Anal. Ghem. 23, 717·20 (~951) .
16 NITRITES, NITRATES, AND NITRO COMPOUNDS 2-NITROFLUORENE By reduction of 2-nitrofluorene to 2-aminofluorene the
16 NITRITES, NITRATES, AND NITRO COMPOUNDS
2-NITROFLUORENE
By
reduction
of
2-nitrofluorene
to
2-aminofluorene
the
color
on
diazotization is suitable for estimation. 82 Alternatively use the color
in acetone solution when made alkaline.
Sample--Tissue. Prepare the extract as described for 2-amino-
fluorene (page 237), but for the development with alkali stop before
"Evaporate to dryness
,,"
Procedure--As 2-aminoftuorene. To an aqueous or acetic acid
solution of sample containing 0.03-0.2 mg. of 2-nitrofluorene add a few
drops of saturated stannous chloride solution. Continue by addition
of water or acetic acid and reagents as described for 2-aminofluorene
(page 000).
With alkaU. Evaporate the acetone extract containing the sample
to 4 ml., or dilute with acetone to that volume. There should be 0.05-
0.5 mg. of 2-nitrofluorene present. Add 2 ml. of 24 per cent potassium
hyroxide solution and mix well. After 15 minutes centrifuge and
read the magenta color of the acetone layer against a reagent blank. '
DINITROBENZENE
Since benzene and nitrobenzene are determined as dinitrobenzene
it follows that dinitrobenzene can be similarly determined,ss The blue
color of dinitrobenzene in anhydrous acetone solution with sodium
hydroxide 84 is stabilized by preparation of the caustic reagent by
solution of sodium monoxide. a6 The same reaction, but with modified
colors, is given by dinitrochlorobenzene, dinitronaphthalene, dinitro-
toluene, and dinitroxylene and has been applied to determination of
varied mononitro derivatives by further nitration. The method will
determine 0.05-0.5 per cent of the compound with accuracy to ±3
per cent.
Procedure--By
sodium
hydroxide in acetone.
Use a sample in
acetone and follow the procedure given for nitrobenzene samples nitrated
to dinitrobenzene (page 12),
82 Benton R. Westfall, J. NatZ. Cancer 11'18t. 6, 23-9 (1945).
83 I. M. Korenman and A. M. Fisher, Zavodikaya Lab. 14, 1058·60 (1948).
8. O. Rudolph, Z. aMZ. Chem. 60, 239-4 0 (1921).
86 F. L. Eniliah, Ana'. Chem. 20, 745-6 (1948).
CHLORO-2,4-DINITROBENZENE 17 By sodium hydroxide in acetone-ethanol. Dissolve 1 gram of sodium monoxide, not sodium
CHLORO-2,4-DINITROBENZENE
17
By sodium hydroxide in acetone-ethanol. Dissolve 1 gram of sodium
monoxide, not sodium peroxide, in 5 ml. of ethanol and 5 ml. of an-
hydrous acetone, each accurate to 5 per cent. Dilute to 100 ml. with
acetone and filter into a dry bottle. The slightly yellowish reagent
must be used within 1 hour.
Dilute 1 gram of sample containing dinitrobenzene to 100 ml. with
acetone. To 25 ml. of reagent add 1 ml. of the sample in acetone. Mix,
read within 30 seconds at 525 mp against the prepared reagent as the
blank, and compare with a calibration curve prepared with the same
substrate and the same isomer or mixture. The color alters quickly.
CHLORO-2,4-DINITROBENZENE
This dinitrochlorobenzene gives a yellow color in anhydrous acetone
and ethanol with sodium hydroxide. s6 The u e of this compound to make
2,4-dinitroanisole makes necessary a method for the former in the presence
of the latter. After reaction with pyridine by heating, a red color in
ethanol is measured. 81 Neither 0- nor p-nitrochlorobenzene reacts. Quino-
line cannot replace pyridine.
Procedure-Air. Pass a known volume of air through absolute
ethanol in suitable absorption bottles. When 8, sufficient concentration of
chlorodinitrobenzene has been built up, dilute the ethanol solution with
four volumes of anhydrous acetone for the use of aliquots. Follow the
procedure for dinitrobenzene by sodium hydroxide in acetone (page 12)
but use a 2-ml. aliquot.
2,4-Dinitroanisole. Mix a 0.5-gram sample, expected to contain
about 0.5 per cent of test substance, with 5 ml. of colorless pyridine.
Swirl and heat at 100 0 for 30 minutes. Cool, dilute to 25 ml. with
ethanol, and mix.
Read at
5 0 mil against
a blank made
with
pure
2,4-dinitroanisole. As a check dilute 1 ml. of the developed solution to
about 20 ml. with ethanol, add 1 ml. of 2 per cent sodium hydroxide
in ethanol, and dilute to volume with ethanol. Mix and read at 580 mil.
As is evident from the dilution, this reaction is much more sensitive.
It is also more reliable.
Compare both results with calibration curve made from 2,4-dinitro-
86 B. A. Rnshkovan, J . Applied Chem. (USSR) 9, 576·9 (1936).
81 E. Voenigricliten, Ber. 32,2571 (1 99); T. Zincke et al. Ann. 330, 361 (1903);
Ibid. 333, 296 (1904); Ibid. 138, 107 (1905); Ib id. 339, 193 (1905); Milton S.
Schechter and H. L. Haller, Ind. Eng. Chem., Aanal. Ed. 16, 326·7 (1944).
18 NITRITES, NITRATES, AND NITRO COMPOUNDS anisole with addition of known amounts of the test
18 NITRITES, NITRATES, AND NITRO COMPOUNDS
anisole with addition of known amounts of the test substance.
reduces the color developed and adds a yellow color.
This
2,4-DINITROTOLUENE
To determine 2,4-dinitrotoluene in air, samples of atmosphere are
absorbed in a naphthalene filter 88 and the subsequent solution of the
naphthalene in acetone and cyclohexanone, followed by addition of
alkali.ao The color develops slowly and is unstable. The wave length is
modified by selection of the solvent mixture, since acetone gives a royal
blue, cyclohexanone a violet blue, and metbylethyl ketone a violet.
To determine dinitrotoluene in liquid samples, follow the procedure
for dinitrobenzene with sodium hydroxide in acetone (paO'e 12) but
read at 610 mp
Procedure-Gas. Use the midget impinger (Vol II, page 64) .
Substitute for the absorption tube a No. 1 sintered glass micro filter
12 mm. diameter and 30 mm. long attached by a rubber connection
to the nozzle of the pump. Prepare the naphthalene filter by pressing
0.4-0.5 gram of ground material on a disc of filter paper resting on the
sintered surface.
Draw the gas through the filter at the rate of approximately 1 stroke
per 5 seconds. Take 30-50 strokes if concentrations are known to be
high; 100-150 if concentrations are low. Loosen the naphthalene filter
with a spatula and dissolve in a 1: 9 mixture of cyclohexanone and
acetone. Shake to dissolve the naphthalene, add 0.5 ml. of 10 per cent
potassium hydroxide solution, and shake again. Ten minutes after the
addition of alkali, read at 610 mil. This reads only 2,4-dinitrotoluene
so that a convenient approximation is to add one-fourth to the figure as
representing total dinitrotoluene.
DINITROXYLENE
Follow the procedure for dinitrobenzene with sodium hydroxide in
acetone (page 12), but for the meta compound or all three isomers
mixed, use a 2-gram sample and for the ortho and para compounds use
4-O'ram samples, reading at 610 mp
88 H. V. A. Briscoe, J. Boy. Soc. 4rt8 88, 104 (1939).
89 W. M. Cumming and W. G. D. Wright, Brit. J . Ind . Med. 2, 83 -5 (1945)_
DINlTROCRESOL 19 DINITRONAPHTHALENE Follow the procedure for dinitrobenzene with sodium hydroxide in acetone (page
DINlTROCRESOL
19
DINITRONAPHTHALENE
Follow the procedure for dinitrobenzene with sodium hydroxide in
acetone (page 12), but use a 5-gram sample and read at 410 m!'.
2,4-DINITROANlSOLE
The reaction of potassium cyanide with metadinitro compounds to
give a red purpuric acid structure 40 is applicable. 41 Sulfur, as from
rubber stoppers, will destroy the color. The color developed by shaking
a metadinitro compound with concentrated sodium hydroxide is usually
too sensitive for this determination but applicable to very small amounts.
Unless 1-chloro-24-dinitrobenzene, which sometimes occurs as an im-
purity, is remov d, it interferes.
Sample-Insecticidal dust. Weigh a sample expected to contain
about 40 mg. of dinitroanlso1e. Stir with five successive portions of
acetone, decanting through a Gooch crucible. Wash the filter until nearly
to volume and dilute to 100 m!. The clear solution may be yellow
because of pyrethrum oleoresins.
Procedure-To a lO-ml. aliquot add 5 ml. of 0.5 per cent potassium
cyanide solution and mix. Read after one hour at 580 mil against an
acetone blank.
DINITROCR.ESOL
The blue color of dinitrocresol with sodium hydroxide in anhydrous
acetone· 2 has been discussed in more detail for dinitrobenzene (page
11) where the procedure is somewhat different. The method is de-
signed to determine 0.001-0.1 per cent dinitrocresol formed in mono-
nitrotoluene. Another form of the procedure determines 3,5-dinitro-
o-cresol in urine.4 s
Procedure-Mononitrotoluene.
Shake a 10-gram sample of mono-
nitrotoluene with 75 ml. of 0.5 per cent sodium hydroxide solution for
40V. Angor, lIfikroohim. Aota 2, 3-8 (1937).
41 Milton S. Schechter and H. L. Hnller, Ind. Eng. Chem., AnaZ. Ed. 16, 325-6
(1944).
42 F. L. English, Anal. Chem. 20, 745-6
(1948);
Cf. Walter Fischer, Z.
anal.
Chem. 112, 91-6 (193
).
4S V. H. Parker, Analyst, 74, 646·7 (1949).
20 NITRITES, NITRATES, AND NITRO COMPOUNDS 1 minute and dilute to 100 ml. with the
20 NITRITES, NITRATES, AND NITRO COMPOUNDS
1 minute and dilute to 100 ml. with the hydroxide solution.
After 5
minutes, filter and read at 410 m{l against the sodium hydroxide solution.
Urine.
Add
5
ml.
of metbylethyl ketone to 5 ml.
of urine,
and
follow
with
2
grams
of
a
9: 1
mixture
of
sodium
chloride-sodium
carbonate.
Shake
vigorously
and
centrifuge.
Read
the
upper layer
against methylethyl ketone at 430 mp
TRINITROBENZENE
The color developed by trinitrobenzene in ethanol with sodium
bydroxide is read in the presence of major amounts of
dinitrobenzene. u
In acetone, both give colors which darken with time. In ethanol, only the
trinitro compound gives a color. Ammonia reacts similarly, but the
color fades quickly. With large excess present, the amount of sodium
hydroxide is not critical.
Sample-Dinitrooenzene. Dissolve 0.1 gram of dried powdered
dinitrobenzene in ethanol and dilute to 50 ml. for the use of aliquots.
Procedure-Measure an aliquot containing 0.1- 1 mg. of
trinitro-
benzene and add 0.5 ml. of 10 per cent sodium hydroxide solution.
Dilute to 50 ml. with ethanol and read at 502 m!1 within 10 minutes
against a reagent blank.
TRINITROTOLUENE
The usual form of trinitrotoluene, designated as a or 2,4,6, is
estimated in aqueous solution after treatment with sulfite and sodium
hydroxide. 4G Sulfite alone gives less color and hydroxide a different one
with a maximum at 460 mp
The procedure is complicated by the
effect of time in converting the trinitrotoluene to a colored complex
which absorbs at a different wave length. Then the color must be read
for each form of the test substance and the values combined. To
complicate the determination further, the color derived from trinitro-
toluene conforms to Beer's law, but that from the colored complex does
not. The colored complex cannot be reduced to the original test
substance. Ammonium picrate may interfere, but can be corrected for,
U M. L. Moss and M. G. Mellon, Ind . Eng. Chem., Anal. Ed. 14,861·2 (1942) .
• ~ C. C. Ruchhoft
and William G. Mechler, Ibid. 17, 430 -4 (1945).
TRINITROTOL UENE 21 after determination by a modifi.ed procedure (page 24). The red- violet color
TRINITROTOL UENE
21
after determination by a modifi.ed procedure (page 24). The red-
violet
color with diethylaminoethanol is also read. 46
In admixture with other nitrated toluenes, sodium bisulfite in acetone
gives a specific color with a-trinitrotoluene. 47 Samples recovered from
air are reduced with titanium trichloride, diazotized, and coupled with
dimethyl-a-naphthylamine. 48
The blue color of sodium hydroxide with dinitrobenzene or dinitro-
toluene in anhydrous acetone is reddish when given by trinitrotoluene. 411
If both are present, the hues are distinctly different but not simple to
separate photometrically .GO The red color is stable for about 30 minutes,
but if cyclohexanone is substituted for acetone, an intense red color
results which is stable for about a week. A disadvantage is that alkali
is only sparingly soluble in cyclohexanone and the two must be
shaken thoroughly before there is any reaction between the test sub-
stance and the alkali. If the cyclohexanone is admixed with methyl-
ethyl ketone, the color produced is intense, permanent, and conforms to
Beer's law. This reaction is also applied to tetryl [2,4,6-trinitrophenyl-
1-methylnitramine] and differs only in hue when given by trinitro-
toluene. It follows that, if both are present, they will interfere.
Semi-permanent tubes containing trinitrotoluene in butanol with
0.1 ml. of 0.56 per cent potassium hydroxide per 10 mi. are useful as
rough standards for comparison against samples in butanol. G1
Samples-Air.
Bubble the sample througb 10 mI. of ic;opropanol
in a midget impinger (Vol. II, page 64) in the course of 20 minutes.
For concentrations less than 0.1 mg. per cubic meter, an even longer
period is necessary.
Dilute to 10 ml. and develop color by reduction,
diazotizing, and coupling with dimethyl-a-naphthylamine.
Wa.ter and sewage.
Use as received or after appropriate dilution and
develop with sulfite and hydroxide.
46 F. H. Goldman and D. E. Rushing, J. Ind. Hyg. Toxicot 25, 164-71 (1943).
47 G. Haltter and H. Winkler, Die Chemie 57, 124-5 (1944).
48 Sherman S. Pinto and John P. Fahy, J. Ind. Hyg. Toxicol. 24, 24-6 (1942);
Sherman S. Pinto and William L. Wilson, Ibid. 25, 381-90 (1943).
"0 K. Kay, Canadian J.
R es.
19B, 86
(1941);
W.
M. Cumming and W. G.
D.
Wright, Brit.
J.
Ind.
Mea.
2,
83 -5
(1945);
I.
M.
Korenman
and
A.
M.
Fisher,
Z a vod87caya Lab. 14, 1058-60
(1948).
GO M. L. Moss and M. G. Mellon, Ind. Eng. Ch.em., Anal. Ed. 14, 861 . 2 (1942).
01 Thomas E. Cone, Jr., U. S. Naval Med. Bull. 41, 219 -20
(1943).
22 NITRITES, NITRATES, AND NITRO COMPOUNDS HexanitroWiphenylamine present. CJ2 Mix 100 ml. of aqueous solution
22 NITRITES, NITRATES, AND NITRO COMPOUNDS
HexanitroWiphenylamine present. CJ2 Mix 100 ml. of aqueous solution
with 20 grams of sodium chloride and 5 ml. of 1: 2 ammonium hydroxide.
Extract with 10, 10, and 10 ml. of chloroform. Save the aqueous layer
for recovery of hexanitrodiphenylamine. Dry the combined extracts
with anhydrous sodium sulfate and evaporate to dryness. Dissolve in
20 ml. of anhydrous acetone and develop with sodium hydroxide in
anhydrous acetone.
Urine. Dilute a suitable aliquot of a 24-hour sample to 7.5 ml. and
use for diazotizing and coupling with N-(l-naphthyl)ethylenediamin.e
dihydrochloride.
Tissue. Extract with trichloracetic acid of suitable concentration,
diazotize the filtrate, and couple with N-(l-naphthyl)ethylenediamine
dihydrochloride.
Alternatively prepare acetone filtrates from 1 part of tissue and 3
parts of absolute acetone. Evaporate the acetone at room temperature,
suspend in 1 per cent bicarbonate solution, and extract with ether. De-
termine by reduction, diazotizing, and coupling with N-(l-naphthyl)-
ethylenediamine dihydrochloride.
Procedure-By SUlfite and hydroxide. Uncolored solutions. In a
50-ml. sample estimated to contain 0.02-1 mg. of trinitrotoluene dissolve
approximately 1 gram of anhydrous sodium sulfite. After 5 minutes
add 1 ml. of 8 per cent sodium hydroxide solu tion and mix. Filter after
10 minutes to remove precipitated calcium salts, etc., and dilute to a
known volume with fresh 0.02 per cent sodium hydroxide-0.06 per cent
sodium sulfite solution . This will fall in the range 1: 4-1: 50 . Dilution
with water would lessen the color. Read at once at 505 mp and every
minute against a distilled water blank until a maximum is reached which
will be within 10 minutes.
Colored solutions. Treat a sample as though uncolored. Also dilute
a filtered sample with 0.03 per cent sodium carbonate solution and read
at once at 460 mp. and 505 mp. Read the sample diluted with sodium
hydroxide-sulfite against a blank of the same degree of aqueous dilution
as the original sample.
For a 1:50 dilq.tion of the colored complex the ppm. is (EjO.29) the
dilution factor. At 1: 5-1: 25 dilution the factor is 0.26. An instrumental
correction may also be required. For the uncomplexed trinitrotoluene,
calculate as usual from the value with the diluted sample as blank.
52 F. Seifert, Yom WIl8S87', 17, 89·92 (1949).
TRINITROPHENOL, PICRIC ACID 23 By sodium hydroxide in anhydrous acetone. Measure out an aliquot of
TRINITROPHENOL, PICRIC ACID
23
By sodium hydroxide in anhydrous acetone.
Measure out an aliquot
of sample in anhydrous acetone containing 0.05-0'.2 mg. of trinitrotoluene.
Dilute to about 5 ml. with anhydrous acetone and add 1.5 ml. of 50 per
cent sodium hydroxide solution. Mix well, dilute to 10 ml. with anhydrous
acetone, and mix again. Filter and, after about 7 minutes, read at 540 mp
against a reagent blank.
By reduction atnd coupling with cUmethyZ-f}-naphthylamine.
As re-
ducing reagent, dissolve 10 ml. of 20 per cent titanous chloride solution
in 10 ml. of concentrated hydrochloric acid and dilute with water to
100 ml. Also dissolve 5 grams of ferric alum in water, add 5 ml. of 1: 5
sulfuric acid, and dilute to 100 ml. By mixing 1 m!. of the ferric reagent
with 0.5 ml. of the titanium solution, 2 drops of 10 per cent potassium
thiocyanate should show the characteristic ferric thiocyanate color.
To a 5-ml. aliquot of sample in isopropanol containing not over 0.2
mg. of trinitrotoluene, add 5 ml. of 1: 10 sulfuric acid and 0.5 ml. of
titanous chloride solution. Heat for 10 minutes in a water hath, remove,
and add 1 ml. of the ferric alum reagent to destroy excess titanous
chloride. Cool to room temperature and add 1 m!. of 0.1 per cent sodium
nitrite solution. Mix and within 15 seconds add 1 ml. of 0.5 per cent
ammonium sul£amate solution. Mix and add within the next 15 seconds
1 ml. of 1 per cent dimcthyl-alpha-naphthylamine in ethanol. After
1 hour, dilute to 15 ml. and read at 530 mp.
With methylethyl ketone in cyclohexanone. See the procedure under
tetryl (page 24).
TRINITR.OPRENOL,
PICRIC
ACID
J
Picric acid as ammonium picrate is determined spectrophotometric-
ally. liS Complications arise when trinitrotoluene is also present. The
color in alkaline solution is intensified if necessary by addition of a
know amount of glucose. Ii '
Sam pl es-Water and sewage.
Filter or centrifuge to remove
tur-
bidity.
Under extreme conditions coagulate with alum, recooonizing that
results at the 10 ppm. level may be 25 per cent low.
Natural hardness is
without effect.
Dilute to under 40 ppm.
liS C. C. Ruchhott and Francis I. Norris, 1M. Eng. Che'TTI
,
.dnaZ. Ed. 18, 480·3
(1946).
II'R. Stohr and F. Scheibl, Mikrochemie tlcr. Mikrochim• .dcta 36/37, 362·5 (1951).
24 NITRITES, NITRATES, AND NITRO COMPOUNDS Procedure--Trinitrotoluene absent. Read the ammonium picrate solution at 460
24 NITRITES, NITRATES, AND NITRO COMPOUNDS
Procedure--Trinitrotoluene absent.
Read the ammonium picrate
solution at 460 mp. or in the ultraviolet at 335 mp
Trinitrotoluene possibly present.
Read at 460 mp. and 505 mp., after
dilution if necessary with 0.03 per cent sodium carbonate. If the ex-
tinction at 460 mp. is only slightly higher than at 505 mp., colored
trinitrotoluene and possibly organic color are indicated rather than
ammonium picrate. If the 460 mp. value is substantially higher than that
at 505 mp., and the latter is significant, both ammonium picrate and
colored trinitrotoluene are probably present. A value at 460 mp., but
none at 505 mp., indicates only ammonium picrate.
If trinitrotoluene is present, carry through the technic for it in
colored solution (page 22). A gain in extin.ction at 505 mIA- indicates
only that the two forms of trinitrotoluene are present. If, after develop-
ment, the value at 505 mp. increases but is lower than the 460 mp. value,
both ammonium picrate and trinitrotoluene are present. If both values
are the same or lower after treatment, ammonium picrate is absent.
If both may be present, mix 50 ml. of diluted sulfite-hydroxide
treated sample with 5 mI. of concentrated hydrochloric acid. Read after
1 minute at 460 mp. and 505 mp
Decolorization indicates both
are
absent. A considerable decrease from 460 mp. which is ammonium picrate
to 505 mp. where ammoniwn picrate shows no absorption in the con-
centration of sample used, indicates ammonium picrate. This 460 mp.
value gives the ammonium picrate by direct ca lculation . The appro-
priate correction can then be applied to the trinitrotoluene values if
necessary.
2,4,6-TRINITROPHENYL-l-METHYLNITRAMINllJ, TETRYL
The color reaction with alkali in anhydrous acetone given by dinitro-
benzene and trinitrotoluene is also given by tetryl. Stability is increased
and other advantages are introduced by use of a less volatile solvent.
The color is a brownish-red with tetryl and of lesser stability than with
trinitrotoluene. 1I11 Suitable atmosphere analysis apparatus is the midget
impinger (Vol. II, page 64).
Procedure liS-As reagent, mix equal volumes of cyclohexanone and
methylethyl ketone and store in a dark bottle.
Place 10 mI.
of this re-
agent in the sampling tube of the midget impinger, attach to the pump,
IIIIW. M. Cumming and W. G. D. Wright, Brit. J. Ind. Med. 2. 83 -5 (1945).
118 The identieal proeedure is appliea.ble to trinitrotoluene.

'

PARATHION 25 and bubble in the atmosphere. Operate at about 1 stroke per 5 seconds.
PARATHION
25
and bubble in the atmosphere. Operate at about 1 stroke per 5 seconds.
Remove the tube, shake with 0.5 ml. of 12 per cent potassium hydroxide
solution until a maximum color is attained, and allow suspended alkali
to settle out for a minute. Read tbe color at an appropriate wave length.
llEXAN ITRODIPIIENYLAMINE,
DlPlORYLAMINE
The solution from wbich trinitrotoluene has been separated is used
for estimation of hexanitrodiphenylamine.
Procedure -Extract the sample (page 22) with 10, 10, and 10 ml.
of ether. Extract the combined ether extracts with 10, 10, and 10 ml.
of saturated aqueous sodium chloride solution.
Dry the ether extracts
with anhydrous sodium sulfate and filter. Dry and bake at 180 0 for an
hour. Cool and take up in about 90 ml. of 1: 100 ammonium hydroxide
solution. Dilute to 100 ml . and read against a reagent blank.
O-O-DIETIIYL-O-p-NITROPHENYL
THIOPHOSPIIATE,
PARATHION
The strongly cbromophoric p-nitrophenyl group in parathion in
ethanol causes a broad and intense absorption band in the near ultra-
violet with a maximum
n ear 274 m,u.57 It is also estimated by reduction
to the amin e, diazotizin rr , and coupling with N-(l-napbtbyl)-etbylene-
diamine dihydrochloride. G8 Aniline and some of its bomologues give
substantially the same color effect.60 Methyl anthranilate will interfere
unless the solution is washed three times with 1: 2.5 hydrochloric acid.
Both parathion and dimethyl parathion are conveniently determined
by saponification to p-nitrophenol with 4 per cent ethanolic sodium
hydroxide . GO Free p-nitrophenol in the product
introduces complica-
tions, necessarily overcome. Other impurities are O-ethyl-O,O-bis(p-ni-
trophenyl )tbiopbosphate, p-nitrophenetol, and trietbyltbiophosphate.
The direct color of the p-nitrophenol i measured. The monoetbylthio.
phosphate is ordinarily present only to a minor extent. The p-nitro-
phenetol produces only traces of p-nitropbenol during hydrolysis and
trietbylthiophosphate does not interfere. If O,O-diethyl-O,o-nitro-
phenyl thiopbosphate is present it is determined by the absorption at
57 Robert C. Birt and J. B. Giselard,
111141. Chem. 23, 185·7 (1951).
58 P. R. Averell and M. V. Norris, Ibid. 20, 753 -6 (1948); J. C. Gage,
75,189 (1950).
4nalyst
59 F. I. Edwards, Jr.,
4nal.
C'hem. 21, 1415-6 (1949).
60 J. A. A. Ketelaar and J. E. Bellingman, Ibid. 23, 646-50 (1951).
26 NITRITES, NITRATES, AND NITRO OOMPOUNDS 450 mp. where it is equal to p-nitrophenol and
26 NITRITES, NITRATES, AND NITRO OOMPOUNDS
450 mp. where it is equal to p-nitrophenol and at 405 mp. where it is only
one-fourth as great.
Sample-Benzene extracts. Pass through a sorption column con-
sisting of 1 part of Hyflo-Supercel to 2 parts of .Atapulgus clay. This
r emoves interfering pigments. Determine by N-(l-naphthyl)-ethylene
diamine dihydrochloride.
Citrus peel. Transfer a 5-gram sample of less than 20-mesh material
to a Babcock cream
bottle.
.Add benzene to wet the sample but not
provide excess, usually 5- 6 mI. Stopper overnight. Add water to about
75 per cent of the volume and agitate mildly to hydrate tbe pulp . After
a few minutes dilute to volume and centrifuge to separate the benzene.
Decant that layer and recentrifuge. Use an aliquot for determination
by N- (l-naphthy) ethylenediamine dibydrochloride.
Citrus oil. Use as is.
Liquid or technical parathion. Add a sample equivalent to 0.05- 0.1
gram of parathion to a few mI. of ethanol. Reflux with 10 mI. of fr esh
4 per cent etbanolic sodium hydroxide for 15 minutes. 0001 and dilute
to 100 ml. with ethanol plus 50 ml. of water . This sample solution is
in 50 per cent ethanol containing about 0.4 p er cent of sodium hydrox-
ide. Determine as p-nitrophenol.
Wettcible powders.
Extract a
sample containin
0.05- 0.1 gram of
parathion with ether.
Evaporate the ether extracts to dryness.
Take
up the residue in a few ml. of ethanol and continue as for parathion
from "Reflux with 10 ml.
of
.
.
.
."
Procedure-Air by ultraviolet absorption. Absorb the contaminant
from an appropriate volume of air in ethanol. Dilute with the same
solvent, if necessary, and read at 274 mp
By N-(l-naphthyl)ethylerlediamine.
General. Dilute an aliquot of
sample in benzene to 10 mI. with benzene. Evaporate to dryness in a
stream of air and take up in 10 mI. of ethanol. Add 10 mI. of water
and 2 ml. of 1: 1 hydrochloric acid. Mix a:p.d add 0.2 gram of zinc
dust. Oover and boil gently for 5 minutes. Filter and dilute to about
40 ml. Add 1 ml. of 0.25 per cent sodium nitrite solution and mix.
After 10 minute add 1 mI. of 2.5 per cent ammonium sulfamate
solution and mix. After another 10 minutes add 2 ml. of 1 per cent
aqueous N-(l-naphthyl)ethylenediamine dihydrochloride and dilute to
50 ml.
.After 10 minutes read at 550 mp. against a reagent blank.
2-NrfRO-1,1-BIS(p-CHLOROPliENYL)ALKANES, DILAN 27 Citrus oil and extracts. Prepare an acid-alcohol solution of 60 ml.
2-NrfRO-1,1-BIS(p-CHLOROPliENYL)ALKANES, DILAN
27
Citrus oil and extracts. Prepare an acid-alcohol solution of 60 ml.
of 99 per cent isopropanol and 5 ml. of concentrated hydrochloric acid
and dilute to 100 ml. with water. Mix 0.4 gram of zinc dust, 0.6 gram
of paraffin, and 4 ml. of this acid-alcohol solution. Add 0.2 ml. of
citrus oil or 1 ml. of the benzene extract of citrus origin. Bring to
boiling within 2 minutes and boil gently for exactly 10 minutes. Cool
in water and filter through cotton. The paraffin will bave solidified
and retain the excess of zinc. Rewarm this with 1 ml. of acid-alcohol
and cool. Filter and wasb with acid-alcohol to total about 9 ml. Add
4 drops of 0.25 per cent sodium nitrite solution, mix, and let stand
for 10 minutes. Add 4 drops of 2.5 per cent ammonium suliamate
solution, mix, and let stand for 3 minutes. Dilute to 10 ml. with acid-
alcohol and remove a 5-ml. aliquot. The residue is a sample blank.
At the end of 3 minutes add 4 drops of 0.1 per cent aqueous N-
(l-naphthyl)ethylenediamine dibydrochloride. After 10 minutes dilute
to 10 ml. with acid-alcohol, filter, and read at 555 mp against the sample
blank similarly diluted.
As p-nitrophcnol. Dilute the sample as p-nitrophenol in alkaline
50 per cent ethanol to 0.3 mg. per 100 ml. with 1 per cent sodium
hydroxide in 50 per cent ethanol. Deviations of ±1 0
per cent in
ethanol or alkali do not affect the color. Read at 405 mil. Corrections
for impurities must be applied.
DIMETHYLPARATIIION
Dimetbylparatbion is determined as p-nitrophenol by the method
described for parathion.
26) from "Reflux with 10 ml
Sample-Dissolve a sample equivalent to 0.05-0.1 gram in a few
ml. of ethanol and proceed as for liquid or technical parathion (page
"
2-NITRO-1,1-BIS(p-CHLOROPIIENYL) ALKANES,
DILA.N
Dilan is a mixture of 2 parts of the butane derivative and 1 part
of the propane compound. By shifting the equilibrium to ~he aci
form
with alkali a complex is formed with ferric ion. 61
Samples-Plant or ammal.
Pulp a sample containing 0.1-2 mg. of
Dilan in water, adding a little hexane if desired.
Extract with 250 ml.
61 Laurence R. Jones and John A. Riddick, Ibid. Z3, 349·51 (1951).
28 NITRITES, Nl'J1RATES, AND NITRO COMPOUNDS of hexane in a Soxhlet for 1 hour. Evaporate
28 NITRITES, Nl'J1RATES, AND NITRO COMPOUNDS
of hexane in a Soxhlet for 1 hour. Evaporate the extract, take up with
ethanol, and dilute to 7 m!. with the same solvent.
Procedure-Fat-free. To the 7-ml. sample in ethanol at once add
1 ml. of 2 per cent sodium hydroxide in m thano!. Mix and after
10 minutes add 3 ru!. of 0.5 per cent ferric chloride in 0.3 N hydrochloric
acid in 50 per cent methanol. Dilute to 12.5 ml. with ethanol and
read at 490 mp against a reagent blank.
Fat present.
If a turbid solution results due to fat,
add
5
ml.
of
ether after the ferric
chloride reagent.
Then
dilute
to
25
m!.
with
ethanol and read at 490 mp against a reagent blank.
CHAPTER 2 ALIPHATIC AMINES AND AMIDES 1 THE classification is such that most primary, secondary,
CHAPTER 2
ALIPHATIC AMINES AND AMIDES 1
THE classification is such that most primary, secondary, and tertiary
amines fall in this chapter unless the primary use is such as to lead
to classification by some other functional group. It follows that many
aromatic compounds come into this classification when the amine
is on a side chain. Some miscellaneous amine derivatives are added.
Methods for hydrazine and hydroxylamine, which are usually con-
sidered as inorganic, are included. Chemically they are amines or
closely related to them.
The general reactions are covered in part as relating to amino
acids, a classification of primary amines following this one. Aside
from that they are diazotizable and develop colors on coupling. Many
develop stable colors with ferric salts . Others are determined by the
color with dimethylaminobenzaldehyde.
ALIPIIATIC
AMTNES
The general reaction of aliphatic amines with sodium 1,2-naptho-
quinone-4-sulionate is applicable to a large number in the absence of
others.2 Applied to amino acids in blood and urine 3 it determines these
as a group with a low degr e of accuracy.4 'fable 2 shows amines to
which the reaction has been applied, including, as a matter of informa-
tion, many to which the reaction is not sati factorily applicable.
The intense color of amine picrates ill solvent is also suitable for
general estimation of
aliphatic amines. 5
The reaction
has been applied
to biological samples 6 and to long-chain amines such as dioctylrnethyl-
1 See Chapter 1, Volume III, for details of organization, condensation, etc.
2 E. G. Schmidt, Ind. Ettg. Cham., Anal. Ed. 11 , 99 · 100 (1939).
8 Otto Folin, J. Biol. Chem. 377-91, 393-4 (1922); Otto Foliu and EiJding
Berglund,
Ibid.
51,
395-418
(1922).
4 N. Howell Furman, George H. Morrison, and Arthur F. Wagner, Ll.nat Chem.
22, 1561-2
(1950 ).
n For more details see determination of trimethylnmine by this method, page 40.
G Derek Richter, Biochem. J.
and Denis Hill, Ib id. 35, 1225-30
32, 1763-9 (1938) j Derek Richter, Ma.rgaret H. Lee,
(1941).
29
30 ALIPHA TIC Al\UNES AND AMIDES amine. 7 It follows that more than one present
30 ALIPHA TIC
Al\UNES
AND AMIDES
amine. 7 It follows that more than one present will interfere with
others.
Thus the reaction determines ephedrine, benzedrine, ,a-phenyl-
ethylamine, mescoline, ,B-phenylisopropylmethylamine, and urotropin. 8
Often differential solubilities can be used for separation.
TABLE 2.
ABSORBANCE
OF
OLUTIONS
OF
2 , 4-DINITROPHENYL
DERIVATIVES
OF
AMINES
Absorption
.Absorbanoe
Maxim1(1fll ml'
Ethanolamine
00409
350
Isopropylamine
0.520
330
",·Hexylamine
••.•
0.507
330
Aniline
• •. •
••.
. •
0.361
335
Benzylamine
•••
0.420
325-330
Dodecylamine
0.326
330
Diethylamlne
.
.
.
0.584
355
Di.n·propylamine
0. 525
355
Di·n,.butylamine
.
0.402
355- 360
Piperidine
.
.
0.423
355
2,3·Dimethylpiperidine
.
0.364
360
Benzyl·N·ethyJamine
Benzyl·N·n,.butylamine
Dibenzylamine
.
0.417
350
0.339
350- 355
.
.
.
.
.
0.193
340- 345
d·Dc8oxyephedrine
0.507
355
N·Methylaniline
0.066
355-360
Diisopropylamine
.
.
.
.
0.000
Dicyclohexylamine
.
.
.
0.000
A solution of 1-fluoro-2,4-dinitrobenzene is applicable as a reagent
with many primary and secondary aliphatic amines.o The absorbancies
of some are given in Table 2 as measured at 3.5 X 10 - 6 M. The basic
reaction is to use excess of the reagent, convert the excess to 2,4-dinitro-
phenol, separate 2,4-dinitrophenylamine from the 2,4-dinitropbenol, and
read the amine. The reaction occurs readily, even with aniline.
Sample-Solutions.
Neutralize the sample solution to just pink with
phenolphthalein and dilute to a concentration of amine or amines being
determined of 0.008-0.025 mg. per ml.
Develop by 1,2-naphthaquinone-
4-sul£onate.
Blood.
To
a
lO-ml.
sample add 1 gram of potassium
carbonate.
Add a drop of 0.1 per cent solution of tyramine to destroy traces of
7 B. M. C. Hopewell and J. E. Page, .Aflalyst 70,17 (1945).
8 C.
Rizzoli, Boll. soo. ital.
biol. spero 25, 433 ·8 (1949).
I) F.
Sanger,
Bioc7lem.
J.
39,
507·15
(1945);
Floyd
C.
Mlllntyre,
Lois
M.
Clemente, and Muriel Sproull, Anal. Chell~. 25, 1757·8 (19r.S).
AMIDES 31 aldehydes. Add 5 ml. of petroleum ether and shake for 30 seconds to
AMIDES
31
aldehydes. Add 5 ml. of petroleum ether and shake for 30 seconds
to remove the amines, the tyramine remaining behind. Discard the
aqueous layer and similarly extract the aroines from the petroleum
ether with 5 ml. of 1: 35 sulfuric acid saturated with sodium bromide .
Discard the petroleum-ether layer and neutralize the acid extract by
dropwise addition of 50 per cent potassium hydroxide solution. Extract
the amines from the prepared and neutralized solution with 3 mI. of
petroleum ether and separate the extract for development by picric acid.
Procedure-By 1,2-naphthoquinone-4-sulfonate. Add the volume of
0.1 saturated sodium carbonate indicated in Table 3 according to the
amine being determined and dilute to 10 ml. Add 2 ml. of fresh 0.5
per cent solution of 1,2-napltthoquinone-4-sulfonate and store in the
dark for 24 hours. Add 2 ml. of a buffer containing equal volumes of
1; 1 acetic acid and 5 per cent sodium acetate. Mix and add 2 m!.
of
4 per cent sodium thiosulfate solution. Dilute to 25 ml. and read. In
the case of mixed aroines or those of unknown structure, it is convenient
to use a dilution of a 0.0534 per cent solution of glycine in 1: 1M
hydrochloric acid containing 0.2 per cent sodium benzoate. Each ml.
is equivalent to 0.1 mg. of amide nitrogen. As indicated in fable 3 the
color match of some amines with a glycine standard is only fair.
By picric acid . To 3 ml. of sample containing 0.0005-0.005 mg. of
test substance add 0.1 ml. of 2 per cent solution of picric acid in
chloroform and dilute to 5 rol. with chloroform. Read after 12 hours
at 445 rop against a r eagent blank.
By 1-jluoro-2,4-dinitrobenzen e. To 0.1 rol. of sample containing
0.01-0.1 mg. of amine add 0.05 ml. of a solution containing 1.2 ml. of
1-fluoro-2,4-dinitrobenzene per 100 ml. of ethanol. Add 0.1 ml. of
0.84 per cent solution of sodium bicarbonate, mix, and heat at 60° for
20 minutes. Add 0.4 ml. of 0.8 per cent sodium hydroxide solution in
60 per cent dioxane and continue at 60° for 60 minutes. Dilute to 10
ml. and add 10 ml. of cyclohexane,
or, in the case of highly water-
soluble amines, use s-tetrachloroethane. Shake and read the organic
solvent extract at the appropriate wave length as shown in Table 3
against a solvent blank.
AMlDES
The reaction of a co centrated solution of an amide with bydroxyl-
amine hydrochloride gi es anN
. e1
of an acethy-
SS&LUP
Regional Ce:l t f(! LI br~ry
Bang"jllr~ )():) 0:4
-
Acces'ion '(\'0
7~
32 ALIPIIATIC AMINES A:r>.rn AMIDES TABLE 3. QUANTITATIVE NATURE OF T HE REAOTION BE'l'WEEN VARIOUS
32 ALIPIIATIC AMINES A:r>.rn AMIDES
TABLE
3.
QUANTITATIVE
NATURE
OF
T HE
REAOTION
BE'l'WEEN
VARIOUS
AMINES AND SODIUM
1,2-NAPRTROQUINONE-4-SULFONATE
Recovery from
0.7 m.q. of Nitro -
0.1 Saturated
Sod1.wm
.qen in 5-mt
SllImvlc witll 01'1-
oine Standard
CoZor Match
Compowna Studied
(m~.)
(%)
with Ocycine
Allylamine
Q-Aminophenol
·m-Aminophenol
p-Aminophenol
Ammonium hydroxide
0.5
100.0
Good
0.5
125.0
Fair. more orange
0.4
154.0
Fair. more orange
0.6
200.0
Fair. more orange
0.4
95.9
Poor, too red
8oc-ButYlll.mine
0.6
50.5
F air. too yellow
Isobutylamine
Cadaverine dihydrochloride .
Di-n-amylamine
Di-n-butylamine
Diisobutylamine
Diethanolamine
Diethylamine
Di-n-propylamine
Diisopropylamine
Ethanolamine
Ethylamine
Ethylenediamine
d-Glucosamule hydrochJoride
Methylamme
Propanolamine
n-Propylamine ••••
lsopropylarrllne •• • •••
Putrescine dihydrochloride
p-Sulfanilic acid
o-Toluidine hydrochloride
Tyramine hydrochloride
0.4
60.6
Poor, too yellow
0.4
99.0
Fair
0.6
102.0
Fair, too orange
0.7
101.1
Fair. too orange
1.0
52.0
Good
0.3
83.3
Good
0.7
97
.6
Fair
.
.
.
0.8
106.1
Fair. too ortlnlle
.
No reaction
0.6
102.9
Good
0.2
50.1
Good
No reaction
0.1
96.4
Good
0.2
100.0
P erfect
0.2
137.0
Fair. more oranlle
0.2
62
.6
Fair, too yellow
.
0.1
47.6
Poor, too yellow
0.5
102.0
Good
0.0
192.3
Fair. m.ore oranlle
1.0
103.1
Good
0.5
111.0
Fair. more red
droxamic acid. 1o This has been developed for determination of a series
of amides shown in 'l'able 4.u The reaction is analogous to the con-
version of esters or anhydrides into hydroxamic acids. The end product
is developed with ferric chloride. The color fades slOWly. Optimum
conditions differ with different compounds as shown in the table. After
the optimum time gradual decomposition of the product occurs. Bar-
biturates vary with the substituent. Betaines do not react.
Procedure--The sample should be in aqueous solution, of a per-
centage cOhCentration approximating 0.01 M.
Prepare tbe reagent by
mixing equal
parts of 16.4 per cent hydroxylamine
sulfate solution
10 C. Hoffmann, Ber. 22, 2854 (1889).
11 Felix Bergmann, Ana!. Chem. 24, 1367-9 (1952).
LIPID AMINE-ALDEHYDES 33 and 14 per cent sodium hydroxide solution. Add 2 ml. of this
LIPID AMINE-ALDEHYDES
33
and 14 per cent sodium hydroxide solution. Add 2 ml. of this to no
more than 1 ml. of sample, and if less than 1 ml. of sample is used
dilute the whole to 3 m!. Hold at the appropriate temperature for the
time specified in Table 4. After formation of the hydroxamic acid
derivative, cool to room temperature and add 1 ml. of 1 : 3 hydrochloric
acid and 1 ml. of 20 per cent ferric ch loride hexahydrate solution in
1: 160 hydrocliloric acid. Read
within 5 minutes at 540 m'_" except for
fluoroacetamide for which use 500 m'_".
TABLE 4.
OPTIMAL
CONDITJONS
FOR
CONVERSION
OF
AMIDES
INTO HYDROXAMIC A OIDS
Readin.Qof
500·570mp.
Name of Compound
Tem·
peratu.re
CO C. )
Reaction
Filter in
T ime.
U1I4tS per
(Min.)
Mioromole
Acetamide
60
120
90
26
480
103
N · Methylacetamid e
Ac etanilid e
N··Acetylsulfanilamidc
Acetylglycine
Fluoracetamid e
Formamide
60
420
57
60
180
70
60
240
70
.
.
60
240
35
26
60
62
26
60
80
60
10
75
Dimethylformamid ('
Succinimide
Ca proJactam
Asparagine
Glutamine
Gluta t hion e
Glycylglycine
Nicotinamide
N' · Methylnicotinamide m et hosulfnte (
Nicotinic acid methylamide
Coramine (I.icotinic ac id dieth ylamide)
Pantothenic acid. calcium salt
Barbitone
Pentobarbitone
Phenobarbitone
.
.
26
240
45
.
.
60
120
85
.
.
60
420
41
60
180
38
.
,
.
.
. •
60
180
35
60
]20
48
60
120
25
26
480
45
I)
26
360
45
.
60
240
30
60
4
0
6
.
.
26
300
89
.
100
45
1.7
.
60
300
1.5
100
120
7.5
Evipan.
sodium
.
100
30
9
LIPID AMINE-ALDEHYDES
The brown color developed by dried eggs in storage is an ether-
soluble amine-aldehyde developed by reaction of an amine fraction with
the cephalin.12 There is a correlation between palatability and either
ultraviolet absorption or fluorescence. On oxidative degradation of
12B. G. Edwards and B. J. Dutton, Ind. Eng. ahem. 37, 1121·2 (1945).
34 ALIPHATIC AMINES AND AMIDES dried egg powder there is a correlation between palatability and
34 ALIPHATIC AMINES AND AMIDES
dried egg powder there is a correlation between palatability and lipid
fluorescence. ls The same sample serves for estimation of carotenoids.
Sample-Dried eggs. This method gives an extract suitable for
estimation both of carotenoids and of lipid-amine aldehydes. Since
the latter is fluorimetric, the reagents require precautions to avoid
extraneous
effects.
.All glassware must be cleaned with bichromate-
sulfuric acid, thimbles ether-extracted for 2 hours, and ether refluxed
and distilled from lead-sodium alloy. Extract 1 gram of dehydrated
egg with anhydrous ether in a micro-Soxhlet for 4 hours. Dilute the
solution to 25 ml. for reading, carotenoids (Vol. III, page 37) and use
for the lipid-amine fluorescence.
Procedure-By fi,uorescerwe. Dilute 1 ml. of the ether extract to
10 ml. with ether and read the fluorescence when excited by the 365
mp. line of mercury, using Coleman :fi]~ers Bl and P C l against a blank
with 0.4 microgram of quinine sulfate per ml. in 1: 360 sulfuric acid
as fluorescence standard.
By ultraviolet absorption. Read at 270 mp. against a reagent blank.
METHYLAMINE
The reaction of monomethylamine in alkaline solution to give a
red color with lactose is appropriate for its estimation.H The color
increases over a long period of time. Ammonia increases the sensitivity
of the readings and should be standardized. Dimethylamine and
trimethylamine in large concentrations inhibit color development.
Urinary pigments do not interfere.
Sample-Urine. If the concentration of monomethylamine is of
the order of 0.1 mg. per mI. , use 0.1 mL without distillation. At 0.01-
0.1 mg. per ml. of methylamine nitrogen, steam-distil 10 ml. in a
microdistillation apparatus in the presence of 5 mI. of 30 per cent sodium
hydroxide. Add a few drops of mineral oil to reduce foaming. Receive
in 10 ml. of 1: 60 hydrochloric acid and, if the acid reaction to nitrazine
paper tends to disappear, add a few drops of 1: 5 hydrochloric acid.
Steam-distil for 10 minutes, then for 1 minute with the acid below the
ll! H. J. Dutton and B. G. Edwards, Ina. Eng. ahem. 37, 1128·6 (1945); 1M.
Eng. Chem., Anal Ea. 18,38·41 (1946).
14 Andrew A. OnnBby and Shirley Johnson, J. Lob. Olin. Mea. 34, 562·5 (1949);
J. Biol. Chem.
187, 711·17
(1950).
1-PHENYL-2-METIIYLAMINOPROPANOL, EPHEDRINE 35 tip of the condenser, and finally for 1 minute without water in
1-PHENYL-2-METIIYLAMINOPROPANOL, EPHEDRINE
35
tip of the condenser, and finally for 1 minute without water in the
condenser.
Neutralize the distillate to pH 6-7, dilute to 50 mI., and use
aliquots.
At less than 0.01 mg. of methylamine nitrogen per mI. acidify 50 m!.
of urine with 1: 1 hydrochloric acid, concentrate on a water bath to
10 ml., and treat as a sampl e containing 0.01-0.1 mg. per ml. but distil
into 10 mI. of 1: 5 hydrochloric acid.
Procedure-Dilute a sample containing 0.01-0.15 mg. of methyl-
amine nitrogen to 6 ml. Add 0.5 ml. of 6.607 per cent ammonium sulfate
solution neutralized to litmus and preserved with chloroform, less
any known correction for ammonia in the sample solution. Add 0.5 ml.
of 3 per cent lactose solution followed by 0.2 m!. of 20 per cent sodium
hydroxide solution. Mix and incubate without evaporation at 56° for
30 minutes.
Read at 540 mp. against a reagent blank.
1-PHENYL-2-METHYLAMINOPRFPANOL,
EPHEDRINE
Ephedrine reacts with ninhydrin in alkaline solution to give a violet
color.1 5 The dev eloped color is extractable with amyl alcohol, thus
giving a good control on the concentration for reading. The color either
disappears on dilution or does not pass readily into the amyl alcohol
layer with emetine, apomorphine, and dilaudid. No color is given by
alypine, aneurine, antipyrine, brucine, bulbocapnine, caffeine, metrazole,
choline, cocaine, colchicine, codeine, cicutine, dicodid, dionine, eucodal,
eupaverine, harmine, heroine, homatropine, hydrastinine, morphine,
narceine, narcotine, ortboform, papaverine, pantocaine, pilocarpine,
pyramidone, stovaine, theobromine, quinine, quinidine, scopalamine,
scuroform, sparteine, strychnine, veratrine, and yohimbine.
Ephedrine oxidized in alkaline solution with hydrogen peroxide
gives a violet color. IS There is no interference by barbital, calcium
chloride, codeine, magnesium chloride, potassium sulfoguaiacolate, terpin
hydrate, or phosphates. Lactose, lanolin, and sugars interfere. This
alkaloid is also determinable by its amine properties in reaction with
picric acid. Precipitation as the reineckate is not complete, preventing
the application of that method. Ephedrine is determined in benzene
by its reaction with picryl chloride.H Determinations agre d with the
16
H. Wachsmuth, J. pharm. Belg. 4, 186 (1949).
16
Antonio Capone, Boll. chim. farm. 90, 465· 72 (1951).
17 L. F. Chlltten and L. I. Pugsl ey, J. Am . Pharm. A880c. 41 , 108· 10 (1952).
36 ALIPIIATIC AMINES .AND AMIDES amounts in commercial products except ointments, as labeled by the
36 ALIPIIATIC AMINES .AND AMIDES
amounts in commercial products except ointments,
as labeled by the
manufacturer.
Other compounds containing the same chemical groups
may interfere.
Sample-Aqueous isotonic sprlllYS and water-soluble jellies. Make a
sample containing 50-100 mg. of ephedrine hydrochloride alkaline to
litmus with 20 per cent sodium hydroxide solution, and add 0.5 ml. in
excess. Extract with six 15-ml. portions of benzene. Combine the
extracts and make up to 100 ml. with benzene. Remove an aliquot
equivalent to 0.4 mg. of ephedrine and make up to 9 ml. with benzene.
Develop with picryl chloride.
Syrups. Proeeed as with isotonic sprays. If emulsions are formed
during the benzene extraction, add 10 mI. of water to a sample con-
taining 50-100 mg. of ephedrine hydrochloride. Make alkaline with
20 per cent sodium hydroxide solution and extract with six l5-ml.
portions of ether or until extraction is complete. Combine the ether
portions and extract with 5 ml. of 10 per cent sulfuric acid and then with
four 5-ml. portions of water. Proceed as for isotonic sprays, from "Malte
.
.
. alkaline to litmus
.
.
"
Capsules.
Add 10 mI.
of water to
20 capsules.
Wash the empty
capsules with another 10 ml. of water, filter, and add the filtrate to
the first solution.
alkaline to litmus
Continue as for isotonic sprays, from "Make
"
Tablets. Powder 20 tablets and dissolve an amount containing 50-
100 mg. of ephedrine hydrochloride in 20 ml. of water and filter. Wash
the filter, and add to the filtrate about 500 mg. of anhydrous sodium
carbonate and sufficient sodium chloride to make a saturated solution.
Proceed as for isotonic sprays, from "Extract with six 15-ml. portions
of benzene."
Oily nose drops. To an amount containing 50-100 mg. of ephedrine
add 10 ml. of ether. Extract with 5 ml. of 10 per cent sulfuric acid and
then with four 5-ml. portions of water. Combine the acid and aqueous
extracts and make alkaline with 6-7 ml. of 20 per cent sodium hydroxide
solution. Continue as for isotonic solutions, from "Extract with six
15-ml. portions of benzene."
OintmentS'. Dissolve an amount containing 10 mg. of ephedrine
in ether, filter, wash the filter and continue as for oily nose drops, from
"
"Extract with 5 ml. of 10 per cent sulfuric acid .
.
DIMETHYLAMINE 37 Procedure- By ninhydrin. Add 1 drop of 1 per cent ninhydrin solution and
DIMETHYLAMINE
37
Procedure- By ninhydrin. Add 1 drop of 1 per cent ninhydrin
solution and 1 drop of 1 per cent sodium carbonate solution to 2 mi. of
the sample solution and warm. Extract the color completely with a
volume of amyl alcohol selected according to the color intensity and
read at 550 mI'.
By peroxide. To a sample containing 1- 15 mg. of ephedrine, add
4 mI. of 16 per cent sodium chloride solution, 0.45 mI. of 0.4 per cent
sodium hydroxide solution, and 7 drops of 30 per cent hydrogen
peroxide. Dilute to 25 mI. and heat in boiling water for 5 minutes.
Cool and read against a reagent blank.
By picryl chloride. To 9 mI. of sample solution in benzene add 1 mI.
of 0.3 per cent picryl chlor ide in ben7.en e. topper and place in a water
bath at 75-77° for 20 minutes. Let stand at room temperature for
3 minutes, then read at 400 mp against a reagent blank.
DIME7l'HYLAMINE
A complex version of the copper thiocarbamate reaction is applied
to dimethylamine. 18 When shaken in aqueous alkaline solution with
a 5 per cent solution of carbon bi. uJfide in benzene and a copper salt,
the amber tint is measured. The solution of carbon bisulfide in benzene
and aqueous solution of dimethylamine do not react unless ammonia
or other material mutually soluble in both phases is present. Am-
monium acetate serves to buffer ammonium ions. The stability of the
color developed is increased by later complete removal of ammonia
from the benzene layer.
Monomethylamine gives a similar color reaction which decreases in
intensity as the alkalinity of the aqueous phase is increased. The
method does not distinguish between dimethylamine and other second-
ary alkylamines. The intensity of the color obeys Beer's law, if the
dimethylamine content is less than 0.0 !) mO'. ill the proc dure given.
No normal urinar y constituents interfere in tb e determination.
This reaction is also applicable to det rmination of the possible
content of up to 3 per cent of dimethylamine in commercial trimethyl-
amine or monomethylamine. 10 Large amounts of monoethanolamine
must be removed to avoid interference. Small amounts do 110t interfere
18 Harry C. Dowden, Biooh6'T11. J. 32, 455-9 (1938).
19 Edward L. Stanley, Hurry Buum, und J e8sie L. Gove, .&nal. Chem. 23,
1779-2 (1951).
38 ALIPHATIC AMINES AND AMIDES as the absorption of the thiocarbamat is at a wave
38 ALIPHATIC AMINES AND AMIDES
as the absorption of the thiocarbamat is at a wave length about 70 lllP.
lower. Methanol equal to the dimethylamine doe not interfere. Re-
covery to ±1 per cent can be expected. The method for trimethylamine
with picric acid (page 40) is also applic-
able to dimethylamine
interfering substance.
in
the
ab ence
of
Sample~Solutions. Neutralize to phe-
nolphthalein and adjust to 0.0006-0.015 mg.
of dimetbylamine per ml.
Un:ne. Distil a mixture of 5 ml. with 50
ml. of water and 10 ml. of 4 per cent sodium
hydroxide solution . Collect 40
ml. of dis-
tillate, dilute to 50 ml., and use an aliquot
as sample.
Trimethylamine.
Weigh 25 ml. of sul-
furic acid of known concentration, uch as
1: 17, and a drop of methyl red indicator.
Add the sample of trimethylamine until a
yel]ow endpoint is reached. Weigh to deter-
mine the sample used. Back titrate to pink
with a drop or two of acid and dilute to a
range of 0.001-0.005 mg. of dimethylamine
per ml.
Monomethylamine.
Weigh 50 ml. of 1:5
bydrochloric acid with a drop of methyl red
indicator. Add monomethylamine until the
solution turns yellow and weigh to get the
sample weight. Back titrate to pink with a
FIG. 3. Apparatus for separa-
tion of dimethylamine and
drop or two of 1: 10 hydrochloric acid and
dilute to 100 ml. Transfer 5 ml. to the flask
of the apparatus shown in Figure 3 and
add
50 ml. of chloroform. Place 10 ml. of
monomethylamine chloroform in the trap, if not already
charged, and reflux for one hour after a precipitate appears. This distils
off the water as an azeotrope boilin
at 56.1 0 , containing 2.5 per cent
of water.20 Thus precipitation of monomethylamine hydrochloride in
the absence of water removes it from reaction. The dimethylamine
20 L. H. Horsley, Ina. Eng. Cham., Anal. Ed. 19, 508·600 (1947).
METHADONE 39 hydrochloride is in the chloroform. Every trace of water must be distilled. Remove
METHADONE
39
hydrochloride is in the chloroform. Every trace of water must be
distilled. Remove the flask, stopper, and let cool to room temperature.
Decant the chloroform solution through glass wool and wash the flask,
precipitate, and funnel with chloroform. Dilute the chloroform solution
to 100 ml. and add a 5-ml. aliquot to 10 m!. of water. Evaporate the
chloroform without substantial distillation of water and use this aqueous
solution as sample.
Procedure-As reagent dissolve 20 grams of ammonium acetate
and 0.2 gram of copper sulfate pentahydrate in 30 ml. of water, add
10 grams of sodium hydroxide in 25 ml. of water and 20 ml. of con-
centrated ammonium hydroxide, and dilute to 100 ml. with water.
To 10 ml. of sample solution, add 1 ml. of reagent and 10 m!.
of 5 per cent carbon bi ulfide in benzene. Warm to 43-48 0 , stopper,
and shake vigorously for 30 seconds. Add 1 ml. of 30 per cent acetic acid
and shake again for 30 seconds. Cool and centrifuge to separate the
solvent layer. Read at 434 mIL within an hour arrainst a reagent blank.
dl-6-DIMETIIYLAMINO-4,4-DIPHENYL-3-HEPTANONE, METHADONE
Methadone, ordinarily the hydrochloride, is estimated by its color
with m-dinitrobenzene in ethano1. 21 It also reacts with most alkaloidal
reagents. 22 Thus as a typical case it is precipitated with methyl
orange at pH 6, the precipitate taken up in chloroform, and that solution
extracted with 1: 15 hydrochloric acid for reading at 490 mp Precipita-
tion with Reinicke's salt from faintly acid solution, filtration, solution
in acetone, and reading at 515 mIL is also applicable.
Procedure-Evaporate a sample containing about 5 mg. of metha-
done to dryness. Take up in about 1 ml. of ethanol and filter to remove
sodium chloride. Wash the residue with not over 1 ml. of ethanol.
To the filtrate add 4 ml. of 1 per cent rn-dinitrobenzene solution in
ethanol and 4 ml. of 4 per cent sodium hydroxide solution. Dilute
to 10 ml. with ethanol Incubate at about 45 0 for 10 minutes and let
stand at room temperature. Read at 500 mp. against a reagent blank
1 hour after mixing.
21 J. S. Faber, Phal'm. WeclobZad
85, 719 · 23 (1950).
22 Bjarne Salvesen, Medd. Norsk
Farm. SeZsloap. 14,1 ·11 (1952).
40 ALIPHATIC AMINES AND AMIDES TRIMETHYLAMINE The yellow color characteri tic of amine picrates in
40 ALIPHATIC AMINES AND AMIDES
TRIMETHYLAMINE
The yellow color characteri tic of amine picrates in toluene solution
is used to estimate trimethylamine in aqueous solution. 28 The method
was developed for estimation of the degree of deterioration of fish.
There is no interference by proteins, glycine, asparagine, cysteine,
cystine, tryptophan, histidine, tyrosine, glutathione, creatine, creatinine,
allantoin, urea, hydroxylam in e, guanine, betaine, choline, and ammonia.
Lecithin from fat interferes by giving a cloud in the developed solvent
but is removed from the sample by extraction. Similar reactions are
given by mono-, di-, trimethyl-, and ethyl-, and propylamine as well as
indole and histamine. The first two are present to only small extents
and the others not at all in fish before they are organoleptically spoiled.
The general rule is that the picrate color is intensified by number and
length of attach ed chains.
The reaction product of trimethylamine with cis-aconitic anhydride
in toluene and acetic acid is violet and suitable for estimation. 24
for fish muscle starting at "At once add 20
Sample-Fish muscle. Mince 5 grams and su pend in 15 ml. of water.
At once add 20 ml. of 10 per cent trichloroacetic acid solution to pre-
cipitate the protein. Centrifuge when well coagulated and use an
aliquot of the supernatant layer for development with picric acid.
Fa,tty fish. Mince 5 grams and suspend in 5 ml. of 1: 100 sulfuric
acid. Extract fat and lecithin by shaking with two successive 10 ml.
portions of petroleum ether. Discard the extracts and neutralize the
aqueous layer by careful addition of a little over 1 m!. of 10 per cent
sodium hydroxide solution. Dilute with water to 15 ml. and proceed as
"
Procedure-B y picric acid. Dilute an aliquot of sample containing
0.002-0.02 mg. of nitrogen as trimethylamine to 4 ml. Shake com-
mercial 37 per cent formaldehyde solution with magnesium carbonate,
filter, and dilute with 3 volumes of water. Add 1 ml. of this, 10 ml.
of toluene, and 3 ml. of 50 per cent potassium carbonate solution.
Stopper with a cellophane-covered cork and shake 30-40 times to
transfer the amines to the toluene layer. Sbake a 5-ml. aliquot of the
23 Derek Richter,
Margaret H. Lee,
Ilnd Denis
Hill,
B iochem.
J.
35,
1225-30
(1941); w. J. Dyer, J. Fiaheries Research Board Can. 6, 3 5 1 ·8 (1945); W. J. Dyer
and Yvone A
Mounsey, Ibid. 6,359 ·67 (1945);
W. J. Dyer, Ibiil. 7, 576·9 (1050).
24B. T. Chromwell, Bioollem. J. 46, 578·81 (1950).
,B.PHENYLETHYLAMINE TYRAMINE 41 solvent layer with 0.3-0.4 gram of anhydrous sodium sulfate and decant into
,B.PHENYLETHYLAMINE TYRAMINE
41
solvent layer with 0.3-0.4 gram of anhydrous sodium sulfate and
decant into 5 mI. of 0.02 per cent picric acid in dry toluene. Read the
color at 410 mil or in the uJtraviolet at 345 mil against a reagent blank.
If free from ammonia the color is stable for a month.
By cis-aconitic anhydride. Shake a sample containing 0.01-0.1 mg.
of trimethylamine and 3 ml. of saturated pota sium carbonate solution
with 10 ml. of toluene for 1 minute. Transfer the toluene solution
and dry with about 0.5 gram of anhydrous sodium sulfate. Add 5 rol.
of the dried solution to 0.1 ml. of 2.5 per cent cis-aconitic anhydride
solution in glacial acetic acid and dilute to 10 ml. with toluene. Heat
for exactly 10 minutes at 65 0 and cool. Read after 15 minutes at 420
mp. against a reagent blank.
,B-PHENYLETHYLAMINE, TYRAMINE
Tyramine may be visualized as decarboxylated tyrosine . As a
phenol
it gives many of the r eactions of tyrosine. Thus it gives the character-
istic color with p-phenyldiazonium sulfonate. 25 Practically any phenolic
body interferes, necessitating separation of the tyramine. 'fhe yellow
color with picric acid is also suitable for estimation in the absence of
other amines giving the reaction. 26 This is
described earlier as a general
reaction (page 29).
A reaction with a-nitroso-,B-naphthol to give a red color which
fades after a few minutes is sensitive to 1 ppm. but only semiquanti-
tative. 27
SampJe--Protein. Hydrolyze a 3-gram sample of vacuum-dried
protein by refiuxing for 24 hours with 40 ml. of water and 90 ml. of
concentrated hydrochloric acid. All-glass apparatus is desirable.
Evaporate the residue to dryness in vacuo in 200 ml. of water. Boil
on a steam bath with 100 ml. of saturated barium hydroxide solution
for 1 hour. This drives off ammonia and precipitates humin. Filter
on a folded filter or a Buchner funnel. Wash the residue with 10 m!.
of saturated barium hydroxide solution. Add 1: 1 sulfuric acid care-
fully until no further precipitation occurs. Digest, filter, and con-
centrate the filtrate to 30 ml. Add 50 mI. of 50 per cent sodium
25 Milton T. Hanke and Karl K. Koessler, J. BioZ. Chem. SO, 235-6!l (1922).
26 Derek Richter, Bioo1lem. J. 32, 1763 -9 (1938); P. Dessi and C. Rizzoli, Boll.
80C. ital. bioI. spec. 24, 1250·4 (1948).
27 P. Muller, Compt. rend. 800. bioZ. 123, 128·30 (1936).
42 ALIPHATIC AMINES AND AMIDES hydroxide solution and centrifuge. Decant the clear supernatant liquid and
42 ALIPHATIC AMINES AND AMIDES
hydroxide solution and centrifuge. Decant the clear supernatant liquid
and wash the residue with a mixture of 5 ml. of 50 per cent sodium
hydroxide solution and 3 mi. of water. Discard the solid residue.
Combine the decantate and washings and extract with 25 ml.,
10 mI., 10 ml., and 10 mI. of amyl alcohol. Discard the aqueous alkaline
solution and combine the amyl alcohol extracts. Extract the amyl alcohol
with two 50 mI. and two 25 ml. portions of 1: 20 sulfuric
acid. The
histamine and tyramine and small amounts of amino acids are in the
sulfuric acid solution. Add hot concentrated barium bydroxide solution
until alkaline. Add 1: 10 sulfuric acid drop by drop until a s!irrht
excess is present. Digest, filter, and wash the residue with 50 mI. of
water. Concentrate the filtrate to 30 mI.
Add 50 ml. of 50 per cent sodium bydroxide solution and extract
the histamine and tyramine with amyl alcohol as before. The amounts
of amino acids extracted are nealigible. Extract from the amyl alcohol
with 1: 20 sulfuric acid as before and precipitate sulfates with barium
hydroxide. Concentrate to a semisolid condition and follow tbe technic
described for tyrosine (page 124), starting at "Take up with 50 mI.
of water and add 600 mI. of an 0.8 per cent solution of silver sulfate."
Following through to the end, re erve the silver precipitate for separate
determination of histamine. The final sample is purified tyramine for
development by p-pbenyldiazonium sulfonate.
Urine. To 10 ml. of urine add 10 per cent sodium hydroxide
solution until distinctly alkaline and extract with 3 mi. of toluene.
Remove the upper layer as completely as feasible and repeat the ex-
traction. Mix the combined extracts, centrifuge to separate water,
and use 3 ml. of the extract as sample for development with picric acid.
Procedure--With p-phenyldiazonium sulfonate. Follow the technic
described for tyrosine (page 126) but use a curve developed from
tyramine.
With picric acid.
Use the general method (page 31).
4,4-TETRAMETHYLDIAMINODIPHENYLMETHANE,
TETRA
BASE
The yellow nitro derivatives formed by the reaction of tetramethyl-
diaminodiphenylmethane with nitrous acid in strongly acid solution
is the basis of a method; most of the yellow color is attributed to
p-nitrodimethylaniline. 28 Any compound that is extracted by bydro-
28 S. Walter Denton, R. M. Oliver, and John T. Wiley, Proc. Am. Petro~6'Um
Ind. 111, 29M, 68·71 (1949).
,B-IMIDAZOLYL-4-ETHYLAMINE, HISTAMINE 43 eblorie acid and forms a '0101' interferes. Thus dimethylaniline
,B-IMIDAZOLYL-4-ETHYLAMINE, HISTAMINE
43
eblorie acid and forms a
'0101' interferes.
Thus dimethylaniline inter-
feres, but aniline, quinoline, pyridine, and p-tolnidine do not. Pbenol
interferes at concentrations above 1 per cent, but is removed by ex-
traction with alkali. The method is consistently about 5 per cent low.
Sample-Oil or grease. Weigh an oil or grease sample containing
1-1.6 mg. of tetra base into 25 ml. of 200 0 -endpoint naphtha. If the
sample contains more than 1 per cent of phenol, extract with 10 ml.
of 0.4 per cent sodium hydroxide solution to remove phenol. Discard
this extract. Extract the naptha solution with two 5-ml. portions of
1: 120 hydrochloric acid, and one 5-ml. portion of water. Combine these
axtracts for use as sample.
Procedur~To the acid solution of sample add 2 ml. of 10 per cent
sodium nitrite solution and 25 ml. of ethanol. Mix and dilute after
5 minutes to 100 ml. with water. Filter if turbid, discarding the first
5 ml. of filtrate. Read immediately at 420 mp, against a reagent blank.
{J-IMIDAZOLYL-4-ETHYLAMlNE,
HISTAMINE
Structurally histamine is decarboxylated histidine. The reaction with
p-phenyldiazonium sulfonate is used for it estimation after interfering
substances have been removed. 20 The method is applicable to 0.01 mg.
of the imidazole. By replacement of the sodium carbonate solution in
the reaction by sodium hydroxide olution, the reaction is less subject
to interference. 3o Accuracy is to ±3 per cent down to 0.001-0.005 mg.
Addition of ethanol after the diazo reagent stabilizes the color for
about an hour. s1 An alternative is the use of p-nitrophenyldiazonium
chloride made by diazotizing p-nitroanilin e. 32 The resulting colored
compound can be extracted from the mixture with methyl isobutyl
ketone. ss The sensitivity is approximately to 0.0005 mg.
29 Karl K. Koessler and Milton T. Hanke, J. Biol. Chc1n. 39,497-519 (1919).
Milton T. Hanke, Ibid. 66, 475 -88 (1925); A. Ma.ciag and R. Schoental, Mi'kro·
ohemic 24, 243-50 (1928); E. Havinga, L. Seckles and Th. Strcngers, Jr., Rec. tra4J.
ohim
66 , 605 -10 (1947); G. Marac, BuU. 800. chim
biol. 32, 287 ·9 (1950) .
80Ryuhei Yokoyama, Japan J. Mea. Sci. VIII , 4, No.1;
Proo. Soc. Internal
Mea. 31 , 208-10 (1936).
31 A. Maciag an.d R. Schoental, Mi'kroohcmic 24, 243-50 (1928).
82 Pietro Dessi and Tullio Franco, Boll. 800. ital.
biol.
8per. 25, 1368· 70 (1949).
3S Sanford M. Rosenthaler and Herbert Tabor, J. Pharmacol. Exptl. Therap.
92, 425·31 (1948).
44 ALIPHATIC AMINES AND AMIDES ITistamine reacts whh 2,4-dinitrofluorobenzene to form a stable colored derivative,
44 ALIPHATIC AMINES AND AMIDES
ITistamine reacts whh 2,4-dinitrofluorobenzene to form a stable
colored derivative, N-a-(2,4-dinitrophenyl)histamine. 1I4
Copper inter-
feres, so that even water free from copper must be used. Phenol should
be pre ent only in very small amount and can be removed by extraction
with benzene. The method is applicable to 0.001-0.008 mg. of histamine
base per m!.
Histamine also gives a violet color in the presence of cobalt nitrate
and sodium hydroxide. 35 The complex is sensitive to oxidizing and
reducing agents. Dissolved air must therefore be removed for accurate
results. Large amounts of ammonium salts interfere. Pilocarpine and
anserine give no color. Imidazole dicarboxylic acid gives a faint rose
precipitate. Imidazole gives a stable violet precipitate. No color is given
by creatinine, tyramine, xanthine, hypoxanthine, and thymine. Guanine
and adenine give a transient violet wbich soon disappears in vacuo.
On extraction of a trichloroacetic acid filtrate witb ether, indole,
purines, and pyrimidine are removed. Histamine is tben extractable
from the aqueous layer with amyl alcobol. Finally, the bistamine is
extractable from the amyl alcohol witb 1: 10 hydrochloric acid. s6 This
is suitable for diazotizing and coupling.
The general color reaction of amino nitrogen compounds with 1,2-
napthoquinone-4-sulfonate in alkaline solution to form bighly-colored
compounds 37 is applicable by the red color with histamine in the
absence of otber amines. as The method is rapid and accurate to
+3
per cent for samples up to 0.08 mg. Glycerin and phenol do not inter-
fere, whereas ammonia and nitrogenous bases, such as the alkaloids
and aminobenzenes, give positive reactions.
Sample-Dry solids. Mix 5 grams witb about 50 ml. of 1: 1 bydro-
chloric acid. Reflux on a sand bath for 30 hours. Evaporate the
acid and water at 60 0 in vacuo and dry in vacuo at not over 80 0 for
1 bour. Dissolve the residue in 75 ml. of water and add 1 ml. of a thick
lime slurry. Add 40 ml. of etbanol and evaporate to 50 ml. in vacuo
84 Floyd C. McIntire, J
.
.Am.
Phwrm.
.AS80C., Sci Ed. 41, 277
(1952).
85 Walther Zimmermann, Z. physicl. Chem.
186, 260·2
(1930).
36 Jacques
Baraud,
Louis
Genevois,
Gabriel
Mandillon
and
Guy
Ringenba.eh,
Compt. rend. 222, 760·1
(1946).
87 Elizabeth G. Frame, Jane A. Russell, and Alfred E. Wilhelmi, J. Biol. Chem.
149, 255·7(} (1943).
88 W. J. Mader, H. S. Sterne, Jr., J. Rosin and H. A. Frediu.ni, J
.
.Am. Pha.rm.
.A8800. 39, 175·6
(1950).
,8-IMIDAZOLYL-4-ETHYLAMlNE, HISTAMINE 45 at 40 0 • T his removes ammonia. To eliminate humins, filter
,8-IMIDAZOLYL-4-ETHYLAMlNE, HISTAMINE
45
at 40 0 • T his removes ammonia. To eliminate humins, filter on a
Buchner funnel and wash the residue thoroughly with hot water.
Add hydrochloric acid to the filtrate until it is strongly acid to
litmus. Evaporate to dryness on a water bath in glass. Dissolve the
residue in about 100 mi. of water, containing 9 mi. of concentrated
hydrochloric acid. Dissolve 20 grams of phosphotungstic acid in 250 ml.
of water containino- 9 mI. of concentrated hydrochloric acid per 100 mi.
Heat the solution of bydrolyzed sample and phosphotungstic acid
solution nearly to boiling and mix. Let cool slowly to room temperature.
Then cool in a refrigerator for 24 hours and filter with suction. Wash
the precipitate with an ice -cold solution containing 18 mi. of con-
centrated hydrochloric acid and 15 grams of phosphotungstic acid in
200 mi. The precipitate contains histamine, histidine, arginine, lysine,
cystine, tyramine, and some other amines.
Suspend the precipitate in 500 mI. of hot water. Add an excess of
a hot saturated solution of barium hydroxide. Heat for 1 hour on a
water bath, cool, and filter on a Buchner funnel. Wash the precipitate
with hot water. lIeat the filtrate on a water bath and precipitate nearly
all of the barium by careful addition of 1: 35 sulfuric acid. Digest and
filter on a folded filter. Evaporate the filtrate to dryness in glass.
Dissolve the residue in the smallest possible volume of water and
dilute to a known volume.
Mix 10 ml. of sample solution with 3 grams of solid sodium
hydroxide and extract 6 times with 20-mi. portions of redistilled a.myl
alcohol. Combine the amyl alcohol extracts and extract 5 times with
10-mI. portions of 1: 35 sulfuric acid.
Heat the acid extracts 011 a water bath and add saturated barium
hydroxide solution until faintly alkaline. Filter while hot and evap-
orate the filtrate to dryness in glass. This residue contains all the
histamine and small amounts of amino acids such as histidine. To
eliminate such amino acids, dissolve the residue in 10 mi. of water
and repeat the extraction exactly as for the original 10 mi. of sample
solution. Finally dissolve the purified residue in water and dilute to
25 mi.
If desired, the histamine fraction may be carri('d throu.,
h
additional
purification steps, which insure greater aCCllracy of the results. Dilute
the solution of purified residue to 100 ml. Add 5 ro]. of 20 per cent
silver nitrate solution. Add 8 grams of barium hydroxide dissolved
in 50 mi. of warm water. Filter on paper by suction. Wash the
precipitate with 50 mI. of cold saturated barium hydroxide solution .
46 ALIPHATIC A~U!\TES AND AMIDES The filtrate must be clear. The histamine is all in
46 ALIPHATIC A~U!\TES AND AMIDES
The filtrate must be clear. The histamine is all in the silver precipitate.
Suspend the silver precipitate in 50 ml. of water. Add 3 ml. of
concentrated hydrochloric acid and sufficient sodium sulfate solution
to precipitate the barium completely. Digest on a water bath for 1
hour and filter. Wash the precipitate with hot water. Neutralize
the filtrate with 10 per cent sodium hydroxide solution and evaporate
to 10 mI. The solution shou ld be only faintly yellow. Dilute to a
known volume, such as 25 ml.
A further purification in chloroform is optional. Evaporate the
liquid to dryness in va'OltO. Add 10 ml. of pure methanol and 0.5 gram
of potassium hydroxide to the residue. Add 200 ml. of redistilled
chloroform and place in a refrigerator for 15 hours. Filter through a
small folded filter. Wa h the precipitate with 200 m!. of hot chloro-
form. Add a few drops of concentrated hydrochloric acid to the
chloroform extracts and evaporate to dryness in vacuo. Dissolve the
residue in 10 m!. of water and l' distil in va01to to remove traces of
methanol. Dissolve in water and dilute to 25 mI. for development with
p-phenyldiazonium sulfate or cobalt nitrate.
Hydrated tissue. Take a sample to give about 5 grams of solids.
Add ethanol to a concentration of about 75 per cent. Add a few drops
of acetic acid and heat on a water bath 1-2 hours to extract free hista-
mine and coagulate proteins. Cool, filter, and wash the residue with
ethanol. Evaporate to dryness on a water bath. Continue with the
residue from filtration and the dried extract, separately, as described
for dry solids.
Blood.
Mix 30 ml. with 50 ml. of 1: 1 hydrochloric acid and proceed
as for dry solids from "Reflux on a sand bath for 30 hours."
Blood serum. Let blood clot in a refrigerator. Centrifuge and
separate 30 ml. of pale yellow serum as sample. Treat as the solution
of dry solids from
Injeotion solutions.
add 1 ml. of a thick lime slurry."
Dilute so that the sample approximates 0.035-
0.055 mg. of hi tamine or an equivalent amount of histamine phosphate
in 5 ml. Develop with 1,2-napbthoquinone-4-sulfonate.
Pharmaceutical produots. Prepare a carbonate buffer of about
pH 10.2 by mixing equal volumes of 10.6 per cent sodium carbonate
solution with 8.4 per cent sodium bicarbonate solution. Prepare a
sample containing 0.02-0.1 mg. of histamine base per rol. in a 1: 10
dilution of the above containing 0.07 per cent of sodium diethyl-
dithiocarbamate. Determine by 2,4-dinitrodifluorobenzene.
,B-nIlD.AZOLYL-4-E'l'IIYL.AMlNE, llISTAl\fINE 47 Procedure-By p-phenyldiazonium sUlfonate. .As reagent, mix
,B-nIlD.AZOLYL-4-E'l'IIYL.AMlNE, llISTAl\fINE
47
Procedure-By p-phenyldiazonium
sUlfonate.
.As reagent, mix 4.5
grams of sulfanilic acid with 45 ml. of concentrated hydrochloric
acid and dilute to 500 ml. The solid dissolves slowly but completely.
Dissolve 22.5 grams of sodium nitrite in water and dilute to 500 mI.
Mix 1.5 ml. each of the sulfanilic acid and sodium nitrite solutions
in an ice bath. After 5 minutes, add 6 ml. more of the sodium nitrite
solution, mix, and let stand in the ice bath for 5 minutes. Dilute to
50 ml. and keep in the icebath.
Do not use for at least 15 minutes
after diluting.
This reagent gives
correct l' sults after 24 hours . When
the reagent is mixed with alkali a pale yellow color gradually develops.
Transfer I-x mi. of water and 5 ml. of a 1.1 per cent solution of pure
sodium carbonate to a comparison tube. Note the time to the second
and add 2 mi. of reagent in 5 seconds. Mix by inclining the tube. Add
x mi. of sample solution, containing 0.0002-0.005 mg. of histamine, ex-
actly 1 minute after the reagent began to mix with the alkali. Mix as
before. Read at 500 mp. against a reagent blank. 39
By 2,4-dinitrojluorobenzene. To 1 mi. of sample solution add 1 mI.
of reagent containing 0.15 mI. of 2,4-dinitrofluorobenzene in 25 mI. of
alcohol The reaO'ent solution should be made fresh each week. Mix,
and after 20 minutes, dilute the sample solution to 20 ml. with 1: 160
hydrochloric acid. Extract the acidified solution with an equal volume
of benzene. Read the aqueous phase at 35 mil against a reagent blank .
By cobalt nitrate. To 1 mI. of sample containing about 2 mg. of hista-
mine per mI. add 3 drops of 1 per cent cobalt nitrate solution. Add
10 drops of 8 per cent sodium hydroxide solution. Mix and read the
color against a rea gent blank . In the absence of histamine, only a faint
blue due to basic cobalt compounds results.
By 1,2-naphthoquinone-4-sulfonate. To 5 mI. of sample add 1 ml. of
1 per cent sodium tetraborate solution. Mix and add 1 ml. of 0.5 per cent
aqueous reagent, less than 8 bours old. Mix and place in boiling water
for 10 minutes. Cool at 4_10° for 5 minutes. Add 1 mI. of a solution
composed of 45 mI. of 1: 10 hydrochloric acid and 10 ml. of 40 per cent
formaldebyde all diluted to 80 ml. with water. Then add 1 mI. of 2.5
per cent sodium thiosulfate solution to decolorize excess reagent. Dilute
to 10 mI., mix, and set a ide for 30 minutes. Read at 460 mfl against a
reagent blank. The factor for conversion of histamine to hi tamine acid
phosphate is 2.76 .
89 Erik Jorpes, B ioohem. J. 26, 1507·11
(1932) .
48 ALIPHATIC AMINES AND AMIDES 3-DIETHYLAMINO-2,2-DlMETHYL TROPATE After separation, 3-diethamino-2,2-dimethyl tropate
48 ALIPHATIC AMINES AND AMIDES
3-DIETHYLAMINO-2,2-DlMETHYL TROPATE
After separation, 3-diethamino-2,2-dimethyl tropate is nitrated and
read. 40
Prooedure--Make 5 ml. of sample, expected to contain 2-3 mg. of
t st substance, alkaline by addition of 1: 1 ammonium hydroxide. Extract
successively with 10, 5, and 5 ml. of ether. Extract the combined ether
extracts successively with 10, 10, and 10 ml. of 1: 160 nitric acid. Dilute
these acid extracts to 50 ml. Mix 2 ml. of the extract with 10 drops of
fuming nitric acid and evaporate to dryne s on a steam bath. Take up
the residue in 2: 98 absolute ethanol and acetone. Dilute to 10 ml. with
the solvent and add 5 drops of 3 per cent potassium hydroxide in
methanol. Read at 570 mp within 5 minutes.
3,4,5-TRIMETHOXYPHENYLETRYLAMINE
The yellow color of picric acid with 3,4,5-trimethoxyphenylethyl-
amine is suitable for reading.41 Follow the general procedure
(page 31).
PROSPRATIDYLETRANOLAMINE, CEPHALIN
The complex phosphate cephalin is hydrolyzed to an ammonium salt
and determined by treatment with sodium hypochlorite. 42
Sample--Nerve
tissue.
Soak a finely
ground
weighed sample
of
nerve tissue for 24 hours with a known volume of ethanol. Centrifuge
and dilute an aliquot to 0.4-1.5 mg. of test substance per mI.
Prooedure--Heat a 1-ml. aliquot of prepared sample with 2 ml.
of 1: 5 hydrochloric acid in a hot water bath for 3 hours. Cool and dry
in vacuo over sodium hydroxide. Extract the residue with 5 mI. of
anbydrous ether to remove fatty acids. Centrifuge and retain the ether-
free residue. This contains the ethanolamine as a salt. Add 6 ml. of
concentrated hydrochloric acid and evaporate to dryness to convert to
an ammonium salt. Take up in 0.5 ml. of water.
As a buffer dissolve 7.32 grams of boric acid and 2 grams of sodium
hydroxide in water and dilute to 1 liter. Add 0.5 ml. of buffer to the
40 Teodor ClUlblick, Fa.rm. Be1J. 45, 665·6 (1946).
41 P. Dessi and C. Rizzoli, Boll. soc. ita.l. biol. speD. 24, 1250·4 (1948).
42 P. V. Edman IUld S. E. G. Aquist, .data Physiol. Bcand. 10, 144·9 (1945).
CORBASIL 49 neutral sample and then 0.5 ml. of 0.5 per cent chlorine water adjusted
CORBASIL
49
neutral sample and then 0.5 ml. of 0.5 per cent chlorine water adjusted
to pH 10 with 4 per cent sodium hydroxide. After this treatment with
sodium hypochlorite, dilute to 5 ml. and heat in boiling water for 5
minutes. Read at 610 mp against a reagent blank.
INDOLE ETHYLAMINE, 3- (2-AMINOETRYL) -INDOLE,
TRYPTAMINE
Tryptamine after isolation gives a distinctive color with either dini-
trobenzaldehyde or dioxane. 4s Alternatively, for determination by ferric
chloride in the presence of trichloroacetic acid, follow the technics for
/3-indolylacetic acid (Vol. III, page 341).
Procedure-Blood serum. Treat the serum with a 1: 1 mixture of
ethanol and acetone containing 0.1 ml. of concentrated hydrochloric acid
p er 100 ml. Filter and evaporate the filtrate to dryness. Take up in
ethanol and dry with excess of solid mer curic acetate. Take up the new
residue in 1: 1 ethanol and water, neutralize, and dry. Di solve the
residue in 10 ml. of 85 per cent phosphoric acid in the presence of 1 ml.
of 1 per cent m-nitrobenzaldehyde or dioxane. Read against a reagent
blank.
PIPElUDINE-/3-CARBOXYLIC
ACID
DIETHYL
AMIDE,
CORAMINE
The familiar reaction with cyanogen bromide and benzidine is applied
to coramine for estimation. 44
Procedure-Dilute
a sample
solution containing 0.25-0.5
mg.
of
coramine to 1 ml. and add 0.5 ml. of a 4 per cent cyanogen bromide
solution. Heat for 1 minute at 90° and add 1 ml. of 0.5 per cent benzidine
in 10 per cent acetic acid. Shake for 5 minutes and dilute to 10 ml.
with 70 per cent ethanol. Read within an hour against a reagent blank.
O-DmYDROXYPIIENYLPROPANOLAMlNE
HYDROCHLORIDE,
CORBAsn.
The method of oxidation of epinephrine with iodine is also applicable
to corbasil. 411 Procaine does not interfere.
Procedure-Dilute
an
aqueous
sample
containing
0.1-1
mg.
of
corbasil to 25 mI. and add 5 ml. of 0.7 per cent solution of disodium
43.A
Lesure, Union pharm. 1939, 122·3.
44
Juan .A. S{rnchez, S C'IItana mild.
(Buenos .Aires)
1944, I, 1269· 73.
411
Knud.
Jackerott, Da'll.'l7c. Tids. Farm. 15, 217 ·35
(1941).
50 ALIPHATIC AMINES AND AMIDES phosphate dodecahydrate and 1 ml. of 1: 25 hydrochloric acid.
50 ALIPHATIC AMINES AND AMIDES
phosphate dodecahydrate and 1 ml. of 1: 25 hydrochloric acid. Mix
and add 1 ml. of 1.27 per cent iodine solution. Mix and at once
add
2 ml. of 2.48 per cent sodium thiosulfate solution. Dilute to 50 ml. and
read at 529 mp against a blank from which the iodine was omitted.
P-PHENYLISOPlWPYLAMINE,
dl-a-MEII'HYLPIIENETHYLAMINE,
BENZEDRINE,
SYMPAMINE, AMPIIETAMINE
Benzedrine is determined in physiological materials by extraction,
distillation of the extract,4.6 and development of a red dye by diazotiza-
tion . 4.7 The method will determine as little as 0.03 mg. of p-phenyliso-
propylamine in 25 grams of sample with an average accuracy of about
±5 per cent. Nicotine and pyridine do not interfere.
The general color of alphatic amines with picric acid is also applied. 4s
The reaction is far from specific and, as originally applied to urine,
gives a substantial reading due to other amines normally present.
Sample-T o 25 grams of blood, urine, or finely minced tissue add
150 ml. of water, shake for 5 minutes, and let stand for 10 minutes
longer. Add 10 ml. of 10 per cent sodium hydroxide solution and shake
for 5
minutes. Add 30 ml. of 10 p er cent sodium tungstate solution,
shake well, then slowly add 30 ml. of 1: 35 sulfuric acid with continuous
shaking. Finally acidify with 1: 1 sulfuric acid to precipitate proteins
and add 5 mt. in excess. Let stand for 15 minutes and filter.
Neutralize the filtrate with 10 per cent sodium hydroxide and add
1 ml. in excess. Steam-distil, collecting 200 ml. of distillate in 10 ml.
of 1: 8 sulfuric acid. Neutralize the distillate with 10 per cent sodium
hydroxide solution and add 1 m!. in excess. Extract by shaking for
1 minute successively with four 30-ml. portions of ether. Wash the
combined ether extracts with 25 ml. of water and discard the washinO's.
Extract the ether solution with three 10-rol. portions of 1 :70 hydro-
chloric acid. Evaporate the combined acid extracts to dryness and take
up the residue with 1 ml. of water for development by a diazo reagent.
For development with picric acid, measure out a sample of solution
containing up to 0.1 mg. of benzedrine. Make distinctly alkaline by
.6 Wm. D. McNally, W. L. Bergman, and J.
F.
Polli, J.
Lab. Olin.
Mell. 32,
913·17
(1947).
47
Karl
R . Beyer
Ilnd
.T.
T.
Skinner,
J . Pll1wmacoZ,
and EzptZ.
Th erap.
63,
419· 32 (1940) .
• 8 Derek .Richter, Lancet 1938, I , 1275 j
P. Dessi, Jlarm. soi.
e
tcc.
(Pavia)
5,
32·8
(1950).
N-PANTOYL-3-PROPmOLAMINE, P ANTHENOL 51 addition of 4 ml. of 8 per ceut sodium hydroxide solution.
N-PANTOYL-3-PROPmOLAMINE, P ANTHENOL
51
addition of 4 ml. of 8 per ceut sodium hydroxide solution.
Agitate the
solution with 6 ml. of petrolcum ether to extract the benzedrine, and
centrifuge.
U e the clear
olvent layer as sample.
Procedure-By diazo reagent. As a diazo reagent, chill 5 ml. of
0.05 per cent p-nitroaniline hydrochloride solution, mix with 1 ml. of
concentrated hydrochloric acid, and chill in an ice-salt mixture for 10
minutes. Add 3 ml. of 0.7 per cent sodium nitrite solution and keep
in the ice-salt mixture for 6 minutes. Dilute to 100 ml. with water at
room-temperature, mix, and cool in the ice-salt mixture for 10 minutes.
This reagent is stable at 0_4 0 for two weeks.
To 1 ml. of sample solution add 5 ml. of diazo reagent and then
5 ml. of 1.1 per cent sodium carbonate solution drop by drop with
,
so lu tion drop'wise with 'tirring and let stand 10 minutes for color
development. Dilute to 40 ml. and mix. Extract the colored compound
with 10 ml. of n-butanol. If necessary, add a small quantity of sodium
chloride to facilitate separation of the two layers. Read at 530 mp
against a reagent blank.
stirrinO'. After 15 minute
add 1 ml. of 10 per cent sodium hydroxide
By picrl:c acid.
To 3 ml. of the benzedrine solution in petroleum
ether, containing not over 0.05 mg. of benzedrine, add 3 ml. of chloro-
form and 0.6 m!. of a 1 per cent solution of picric acid in toluene. Let
stand for 12 hours for foreign picrates to precipitate and read at 445 mp.
against a reagent blank.
N -PANTOYL-3-PROPANOLAMlNE, P ANTHENOL
Pantbenol is a,y-dibydroxy-fJ,fJ-dimethylbutyramide, one of the vita-
min B complex. By cleavage to fJ-alanine the latter is estimated by
reaction to give an oran"'e-red color with 1,2-naphthoquinone-4-sodium
sulfonate 49 or 2,4-dinitrophenylhydrazine. 5o Cleavage of panthenol and
pantothenates in acid media yields pantoyl lactone and fJ-alanol or
fJ-alanin6 resp ctively. The pantoyl lactone forms its hydroxamic acid
with hydroxylamine in alkaline solution. This gives a purple complex
with
ferric chloride in acid solution. 51 The color starts to fade after a
49 R. Crokuert, Arch. intern. physioZ. 56, No.2, 189·91 (1948).
50 C. R. Szalkowski, W. J. Mader, and H. A. Frediani, CcreaZ Chem. 28, 218·25
(1951).
51 Fritz
Feigl,
V.
Anger,
and
O.
Frehden,
Mikroohemie
15~.22 _l1934) ;
c:>,
S.
Hestrin,
J.
Biot.
Chent.
180,
249·61
(1949);
Ernest
o~~-ttnt~r-t9~
Sclunuli,
Anal.
Chem.
22,
1033·7
(1950).
./ 'f\)~ ~-~--""![I , ,
f' ''y ''--
r't(
N).···
52 ALIPHATIC AMINES AND AMIDES minute. The average deviation is ± 3 per cent. There
52 ALIPHATIC AMINES AND AMIDES
minute. The average deviation is ± 3 per cent. There is no interference
from thiamine hydrochloride, riboflavin, niacinamide, biotin, or folic
acid before or after hydrolysis. Pyridoxine hydrochloride gives a slight
brownish color before or after hydrolysis, corrected by the blank. Ascor-
bic acid interferes. By sorption on resin, panthenol is separated from
panthenates. Also consult the next topic, pantothenic acid, for further
details.
Sample-Water-SOl1l-ble multivitamin preparations. Place plugs of
fiber glass over the stopcocks of three 14 X 200 mID. columns and fill
to 40 mm. with Amberlite IRA-400-0H (Rohm and Haas). Wash each
column with about 50 ml. of 1:4 hydrochloric acid and then with about
25 ml. of water . Add 50 ml. of 10 per cent sodium hydroxide solution,
withdraw it, and wash thoroughly with about 50 ml. of water. Add
water so that about 10 ml. is above the resin and stir to remove bubbles.
Put a plug of fiber glass on top of each column and drain the water
to about 5 rom. above this plug. The columns may be reused by similar
treatment.
Add an aliquot of solution, not over 3 ml., equivalent to 2-3 mg. of
pantbenol to each of 3 columns. To the second column add 1 m!. of
standard containing 1 mg. of panthenol and, to the third, 2 ml. of the
same standard. Allow to flow through the columns at 0.5 ml. per minute
until no liquid remains on top. Then elute with 80 ml. of water at 3 m!.
per minute. Evaporate the eluates to 10 ml. on steam baths with an
air stream and develop. Any pantoyl lactone or pantotbenic acid is
retained by the column.
Oil-ba·se multivitOmtin capsules. Combine capsules containing about
40 mg. of panthenol by cutting with a razor blade. Add 50 mI. of
petroleum ether and exactly 25 ml. of water. Shake mechanically for
1 hour. Separate the aqueous layer and centrifuge for 5 minutes. Filtet:
10-15 ml. of the aqueous layer and use 2-ml. aliquots by the method for
water-soluble preparations.
Procedure-Dilute a sample containing about 3 mg. of panthenol
or pantothenate to 5 m!. with water, or use the prepared sample which
may total 10 mI. Add 3 m!. of 1: 10 bydrochloric acid, stopper loosely,
and heat at 80° for 3 bours. Cool and add 2 m!. of a fresh 7.5 per cent
solution of hydroxylamine in 4 per cent sodium hydroxide solution.
Mter 5 minutes add 3 drops of a 0.1 per cent solution of 2,4-dinitro-
phenol in etbanol as indicator. Titrate to colorless with 1: 10 hydro-
PANTOTHENIC ACID 53 chloric acid and dilute to 50 m!. Mix 5 ml. with 1
PANTOTHENIC ACID
53
chloric acid and dilute to 50 m!. Mix 5 ml. with 1 ml. of 2 per cent
ferric chloride solution and read 45-60 seconds later at 500 mIL against
water. Subtract a blank prepared without hydrolysis.
For the standard described under sample, calculate by (A. X D) /
[(B + 0)
-
2A.
x E] = mg. of panthenol per m!. of sample solution
in which
A. = reading of sample solution
B =reading of sample plus 1 m!. of panthenol standard
o = reading of sample plus 2 m!. of panthenol standard
D = mg. of panthenol added to Band 0
E = ml. aliquot of sample solution used.
PANTOTHENIC
ACID
Pantothenic acid is discussed in part under the preceding topic,
panthenol. As a treatment after cleavage,52 oxidize the fi-alanine with
potassium permanganate in the presence of potassium bromide to give
an insoluble hydrazone with 2,4-dinitrophenylhydrazine. To estimate the
latter dissolve in pyridine and dilute with aqueous sodium hydroxide.
There is no interference by acetic, lactic, tartaric, glycolic, and succinic
acids, a-alanine, ethanol, or riboflavin. Compounds which do interfere
are carbohydrates, ascorbic acid, thiamine hydrochloride, pyridoxine
hydrochloride, niacin, niacinamide, and soya flour. AU of the inter -
ferences except ascorbic acid are eliminated by chromatographing.
Sample-Feed enrichment mixt1~res. Shake a weighed amount of
sample containing about 200 mg. of calcium pantothenate with 200 mI.
of water for 10 minutes. Dilute to 250 ml., mix thoroughly, and centri-
fuge. Prepare a 12 X 500 mm. chromatographic column with 8 grams of
aluminum oxide and activate just before use by washing with 50 mI. of
1: 1 hydrochloric acid followed by 100 mI. of water.
Pass an aliquot containing about 25 mg. of calcium pantothenate
through the column and wash with successive portions of water, totaling
100 ml. Elute the column with 50 m!. of 1: 1 sulfuric acid and then
10 mI. of water. Reflux the combined eluate and washings for 1 hour,
cool to room temperature, and dilute to 250 mI. with water for develop-
ment with pyridine.
52 O. R. Szalkowski, W. J. Mader and H. A. Frediani, Cereal Chern. 28, 218-25
(1951) .
54 ALIPHATIC AMINES AND AMIDES Procedure-To a 25-ml. a.liquot of acid hydrolyzate in about 40
54 ALIPHATIC AMINES AND AMIDES
Procedure-To a 25-ml. a.liquot of acid hydrolyzate in about 40
per cent sulfuric acid, add 5 mI. of 1: 1 sulfuric acid and adjust the
tempera.ture to 22-25°. Add 5 mI. of 12 per cent pota sium bromide
solution and 10 mI. of 5 per cent pota sium permanganate solution.
Leave at 22-25° for 10 minutes, then cool in an ice bath for 5 minutes.
Add 20 per cent sodium sulfite solution dropwise to decolorize the potas-
sium permanganate. Add 10 ml. of 0.5 per cent 2,4-dinitrophenylbydra-
zine in 1:4 hydrochloric acid to tbe clear, colorless solution. Mix the
precipitate and olution thoroughly, heat on a steam bath for at least
15 minutes, and cool to room temperature. Transfer the yellow precipi-
tate to a sintered glass filter and wash with five 5-m!' portions of water.
Dry at 100° for 30 minutes. Meanwhile, drain the precipitation flask
and take up the traces of precipitate in two 3-ml. portions of hot pyridine.
Transfer the pyridine washings to a 25-ml. flask. Fit the sintered o-lass
filter to a filtration flask containing the 25-ml. flask. Add small portions
of boiling pyridine to the filter and triturate the contents with a glass
rod. Use suction to draw the resultant pyridine solution through into
the flask. Dilute to 25 ml.
To a 5-ml. aliquot add 50 mI. of water and 5 ml. of 20 per cent
sodium hydroxide solution. Dilute to 100 ml. and read at 570 mIL against
a reagent blank. The color is stable for an hour.
HYDRAZINE
The orange of the azine formed by hydrazine with p-dimetbylamino-
benzaldehyde is a stable reaction product 53 which is soluble in mineral
acids and develops the maximum color within 15 minutes. Many primary
aromatic amines interfere. Urea, semicarbazide, and adrenaline give a
slight reaction. Ammonium ion does not. The optimum range is at
0.006-0.047 mg. per 100 ml. of sample. Error is under ±1 per cent.
The color develops at once, is stable after 10 minutes, and does not
change for 12 hours. Hydrochloric acid over 1: 15 increases the intensity
of absorption.
Procedure-To
5
ml.
of sample
containing
0.0001-0.001
mg.
of
hydrazine per ml., add 1 mI. of reagent containing 0.4 gram of dimethyl-
aminobenzaldehyde, 20 mI. of absolute ethanol, and 2 mI. of concentrated
hydrochloric acid. Read after 15 minutes at 458 lUl-l against a reagent
blank.
G3 M. PC8CZ and A. Petit, Bull. 800. cMm. FraMe 1947 , 122 · 3; George W. Watt
and Joseph D. Crisp, 41l4l. OMm. 24, 2006-8
(1952).
NEOANTERGAN MALEATE 55 HYDROXYLAMINE Hydroxylamine reacts with benzoyl chloride to give benzylhydrox- amic acid. This
NEOANTERGAN MALEATE
55
HYDROXYLAMINE
Hydroxylamine reacts with benzoyl chloride to give benzylhydrox-
amic acid. This forms a reddish-violet ferric salt in neutal or slightly
acid solution.~4 Substances which react with hydroxylamine in the cold
or which react with ferric ion must be absent. Glucose does not interefere
if its concentration is not more than twenty times that of the hydroxyla-
mine. With a sample concentration of 0.2-1 mg. the error is ±2
per cent. The blue with chlorinated urea is extracted for estimation.lil>
Sample-Solutions. Add 1:1 hydrochloric acid or 10 per cent
sodium hydroxide to a 10-ml. sample with cooling until the color of
phenolphthalein is just discharged. Dilute to a known volume at which
it will contain 0.2-1 mg. of hydroxylamine per ml., expressed as the
hydrochloride.
Procedure-By benzoyl chlor1"de and ferric chloride. To 4 ml. of
sample solution add 2 drops of colorless benzoyl chloride, followed by
4 ml. of ethanol and 2 ml. of a 2 per cent solution of sodium acetate
trihydrate. Add 2 ml. of a 0.5 per cent solution of ferric chloride hexa-
hydrate in 1: 50 hydrochl oric acid and dilute to a known volume. Allow
to stand for several minutes so that undissolved benzoyl chloride will
settle. Read against a reagent blank.
By chlon:nated urea·. Prepare the reagent by saturating 60 grams
of urea in 15 ml. of water with 30-32. grams of chlorine. The mixture of
syrup and crystals decomposes slowly at 20 0 but can be kept for
several weeks. To a lO-Jul. sample add 2 ml. of 10 per cent sodium
acetate trihydrate solution and two drops of cyclopentanone. Mix well
and add 1 ml. of a fresh 10 per cent solution of the reagent. Shake and
add 5 ml. of xylene. When the blue color is extracted, read the upper
layer against a reagent blank.
N-(a-PYRIDYL)-N-(p-METIIOXYBENZYL)-N',N'-DIMETHYLETHYLENE-
DIAMINE,
PYRANISAMINE MALEATE,
NEOANTERGAN
MALEATE
Pyranisamine maleate is a nonproprietary antihistamine most satis-
factorily read directly at 243.5 m,u.!i6 A concentration of 2 mg. per
54 Eug. Bamberger, Ber. 32, 1805·6 (1899);
George W. Pucher and Harold A.
Day, J. Amer. Che'l1~ Soo. 48, 672·6 (1926).
55 O. Wichterle and
M.
Hudlieky,
Collection Cllcohoslav.
Chem.
CO'l1ununs.
12,
661·71
(1947).
~6 Louise
Smith, J. Am.
T.
Anderson,
W.
O.
Gakenheimer,
Oharles
Rosenblum
and
E.
H.
Pharm.
.ds.qoc. 38, 373·7
(1949).
56 ALIPHATIC Al\fiNES AND AMIDES 100 m!. is appropriate. Procedure-TabLets. Allow 0.5 gram of tablets
56 ALIPHATIC Al\fiNES AND AMIDES
100
m!. is appropriate.
Procedure-TabLets.
Allow 0.5 gram of tablets to disintegrate in
100
ml. of water and dilute to 500 ml.
Mix thoroughly, dilute a 10-m!.
aliquot to 500 m!. with water, and read at 243.5 mp. interpreting from
a
standard in water.
Elixirs. Dilute with 20 per cent ethanol or water so that the content
is
2 mg. of maleate per 100 ml. of solution. If there is interference with
direct reading, pretreat another sample as follows. Dilute 10 ml. of
elixir to 100 ml. and extract three times with 10-ml. portions of ethyl
ether. Dilute the aqueous phase to a concentration of 2 mg. of the anti-
histamine per 100 ml. and read at 243.5 mp
Anhydrous petrolatum ointments.
Dissolve 5 grams of ointment in
100 ml. of petroleum ether and extract five times with 25-ml. portions of
water. Combine the extracts, dilute to 500 ml. with water, and mix
thoroughly. Dilute a 10-ml. aliquot to 250 ml. with water and read at
243.5 mp
Anhydrous water-soluble ointments.
Dissolve a 5-gram sample in
water to make 500 ml. of solution. Dilute a 10-m!. aliquot to 250 ml. to
approximate 2 mg. of antihistamine per 100 ml. Read at 243.5 mp.
against the blank containing the ointment base.
Aqueous
em1tlsion-type
ointments.
Dissolve and dilute a 5-gram
sample as described for water-soluble ointment but use equal volumes
of absolute ethanol and petroleum ether as solvent. Read against a blank
at 243.5 mp
Parenteral solutions.
Dilute to approximate 2 mg. per 100 ml. and
read at 243.5 mp
HEXAMETHYLElNETETRAMINE, UROTROPlNE, METHENAMINE
Urotroprine can be converted back to formaldehyde and so deter-
mined. Retention at a proper pH will give the result, but distillation
is preferable. While any formaldehyde method would appear suitable,
those with phloroglucinol 57 and chromotropic acid 58 have been used.
117 R. J. Collins and P. J. Hanzlik, J. BioI. Chem. 25, 231·7 (1916); Alfred T.
Shobl and Clyde L. Deming, J. Urology
4, 419 ·37
(192 0).
58 Fernando Montequi Diaz de Plaze, Aoole8 rea~ 800. eKPMi. fta. '!I qu{m. 4"7B ,
135·42 (1951).
GLUCOSAMINE 57 Procedure-Urine. Distil a measured volume of urine until half the original volume is
GLUCOSAMINE
57
Procedure-Urine. Distil a measured volume of urine until half
the original volume is present as distillate. This contains both the
formaldehyde originally present as such and the hexamethylenetetramine
converted to formaldehyde.
Determine the formaldehyde in the original sample by phloroglucinol
(Vol. III, page 258). Also use the distillate as a ample for estimation
of formaldehyde. Subtract the free formaldehyde from the total formal-
dehyde given by this method and multiply by 0.781 to give the value of
the balance in terms of hexamethylenetetramine.
Medicinals. Determine by chromotropic acid (Vol. III, page 259) .
GLUCOSAMINE
Glucosamine acetate gives a red color with p-dimethylaminobenzalde-
hyde. G9 Mucin, after a short period of heating with dilute alkali, on
acetylation shows the reaction due to the acetylated glucosamine present.
The color which develops fades rapidly. Interference by urea, phosphate,
and other urinary ingredients is negligible. Some sugars interfere, but
the optimum for reaction of glucosamine is pH 9.5, while that for the
sugars is 10.8-11.2.°0 Also glucosamine after heating in sodium carbonate
solution no longer gives the red color with the reagent. The reaction
will detect 0.05 mg. of glucosamine and is acc urate to ±5 per cent on
a sample of 0.5-5 mg.
Another method of application of the same reaction is by forming
a pyrrol derivativ e. 61 Around pH
9.8, small changes have little effect
on the stability of the color formed. 02 This is equally applicable to
solutions of chondrosamine which give the same intensity of the reddish
color as glucosamine. Glucose, galactose, fructose, arabinose, glycine,
alanin e, urea, glutamic acid, proline, arginine hydrochloride, lysine,
serine, phenylalanine, leucin e, tyrosine, hydroxyproline, valine, nor-
leucine, and histidine do not interfere. Some pyrrol or indole deriva-
50 Fritz Zuckerkandl and Luise Messiner·Klebermass, Biochem. Z. 236, 19·28
(1931); Torazo Miyazaki, J. Biochem. (Japan) W, 211·22 (1934); Walter T. J.
Morgan and Leslie A. Elson, Biochem. J. 28, 988·95 (1934).
60 J. Immers alld E. Vass ur, Natwre 165, 898·9 (1950).
61 Leslie A
Elson and Walter T.
J. Morgan, Bioohem
J. 27, 1824·8 (1933);
Ivar Nilsson, Bioohem. Z. 285, 386·9 (1936); John W. Palmer, Elizabeth M.
Smyth, and Karl Meyer, J. BioI. Chem. 119, 491·500 (1937); Hajime Masamune
and Yoshio Nagazumi, J. Bioohll'll~. (Japan) 26, 223·32 (1937); Gunnar Bllx, Acta
Chem.
Eoana.
2, 467·73
(1948).
62 Benjamin Schloss, Anal. Chem. 23, 1321·5
(1951).
58 ALIPllATIC AMINES AND AMIDES tives interfere, but these give the color with the reagent
58 ALIPllATIC AMINES AND AMIDES
tives interfere, but these give the color with the reagent in acid solution
without prior treatment with acetylacetone. The color with tryptophan
does not develop in the concentration of hydrochloric acid used, only
in stronger acid. Many amino acids and carbohydrates also react at
higher acidity. n-Acetylglucosamine or n-acetylchondrosamine react
with the reagent to give about one-fifth the color intensity with a
maximum at 530 mft. The color with pyrrol has its maximum at 550
mft. Variations in pH are critical. Substantially the same color is given
by l-amino-glucose, but this is not likely to be present in acid hydrolysis
products of glucoproteins or nitrogenous polysaccharides. Results are
accurate to ±0.5 per cent if the sample contains 0.012-0 .83 mg. of
glucosamine per m!. Prior treatment with acetylacetone can be replaced
by treatment with ethyl acetoacetate, but the former causes a deeper
color.
Hexosamines, deaminated by nitrous acid, yield 2,5-anhydrohexoses
which, when reacted with indole in dilute hydrochloric acid, give char-
acteristic colors.o8 A correction must be applied for the color developed
with the sample before deamination. If the reaction mixture is shaken
with chloroform, a pink color is taken up, and a brown color remains in
the water phase. However, there is little difference in their absorption
curves, and so separation is of little value. Nitrogen-free carbohydrates
react with indole and hydrochloric acid and give a maximum absorption
at 492 mft, but this is much lower than that of deaminated hexosamines.
Serum albumin at 0.2 per cent and ascorbic acid at 0.01 per cent, when
treated with nitrous acid, show an absorption at 492 mft in the indole
reaction. Hence to avoid interference from these substances, readings
are made at 520 and 492 mft. The increa e of tbis difference after
deamination is a measure of the amount of hexosamine, as serum albumin
and ascorbic acid do not show such an increase. Glucose and glucuronic
acid do not interfere.
Hexosamines occur in polysaccharides in the form of their acetyl
derivatives and determination of these hexosamines must be preceded
by deacetylation by heating for 1 hour with 1: 10 hydrochloric acid.
At 100°, buffered at pH 5.9, glucosamine reduces mercuric chloride
rapidly and the acetate hardly reacts. Buffered at 7.4 glucosamine
acetate reduces the reagent readily.o4 For quantitative application the
68 Zacharias Dische and Ellen Borenfreund, J. Biol. Chem. 184, 517-22 (1950).
64L. Hahn, .drki1J. Kemi. Mineral Geol. An, No. 12,10 pp. (1946).
GLUCOSAMINE 59 mercuric chloride unreduced is determined with diphenylcarbizone (Vol. II, page 73). An alternative
GLUCOSAMINE
59
mercuric chloride unreduced is determined with diphenylcarbizone (Vol.
II, page 73).
An alternative is to add saturated aqueous trisodium phosphate
solution, saturated thereafter with borax. 65 This hydrolyzes the amine
to ammonia for di tillation into dilute boric acid solution and subsequent
estimation by Nessler's reagent.
Samples-Casein. Hydrolyze 1 gram of casein by heating with an
excess of 1: 3.6 hydrochloric acid for 8 hours and evaporate to dryne s
in va.cuo. Dissolve the residue in 50 ml. of water and add 1 gram of
activated carbon. Heat to boiling and filter. Wash the residue thor-
oughly with hot water. Cool the filtrate and dilute to 100 ml. for the
development of aliquots as the pyrrol derivative.
Proteins. Reflux 1 gram with 10 ml. of 1: 1 hydrochloric acid for
5 hours. Neutralize with sodium carbonate and add 20 per cent of the
volume of 20 per cent lead acetate solution. Dilute to 50 ml. Filter and
precipitate lead from the filtrate with hydrogen sulfide. Extract with
ether to remove acetic acid and develop an aliquot as the pyrrol
derivative.
Ovom~,coid. Heat 1 gram of mucin for 3 hours in 1: 4 hydrochloric
acid on a water bath and complete as for casein, starting at
evaporate to dryness in vacuo."
Alternatively, let the sample stand for 2 days at room temperature
in concentrated hydrochloric acid and dilute with 3 volumes of wate·r.
Complete as for casein, starting at"
evaporate to dryness in vac'Uo,"
but omit the carbon treatment.
Blood. Lake 2 ml. of oxalated blood with 2 ml. of water. Shake with
16 ml. of 4 per cent trichloroacetic acid solution to deproteinize. Let
stand for 10 minutes and filter. Extract the filtrate with several suc-
cessive 5-ml. portions of ether, discarding the extracts. Remove ether
from the remaining aqueous solution by bubbling air through it and
use as sample for development as the acetate.
Tissue. Grind 0.2-0.6 gram of tissue in a mortar. Transfer with
4·5 ml. of water, add 10 ml. of 7 per cent trichloroacetic acid solution,
and dilute to 20 ml. Filter after 1 hour. Continue as for deproteinized
blood, starting at "Extract the filtrate
" In the procedure evaporate
to 0.5 ml. by heat and th'11 continue to dryness in va,cllo over phosphorus
pentoxide.
65 M. V. Tracy, Biochem. J. 52, 265-7
(1952).
60 ALIPHATIC AMINES AND AMIDES Urine. To prepare urease, digest 1 part of soybean meal
60 ALIPHATIC AMINES AND AMIDES
Urine. To prepare urease, digest 1 part of soybean meal with 5 parts
of water at room temperature for 1 hour, stirring occasionally. Filter
or centrifuge and evaporate the extract to dryness at less than I-mm.
pressure. Powder the residue. Alternatively pour the extract into suffi-
cient acetone so that it is dehydrated practically instantaneously, filter,
and dry.
If alkaline, acidify with a few drops of 15 per cent trichloroacetic
acid solution. Dilute 1 ml. to 7 ml. Add 0.3 gl'am of urease and 3 ml.
of 4 per cent acid potassium phthalate solution. Heat at 40° for 1 hour
to decompose urea. Add 1 ml. of 1: 10 hydrochloric acid and 9 ml. of
10 per cent lead acetate solution. Mix and filter. Saturate the filtrate
with hydrogen sulfide and filter off the lead sulfide on a Buchner funnel.
Aerate to expel hydrogen sulfi.de and extract with several successive
portions of ether to remove phthalic acid. Aerate to remove ether from
the aqueous layer and use it as sample for development as the acetate.
In the procedure evaporate the volume used to 0.5 ml. and dry in vacuo
over phosphorus pentoxide.
Alternativel y use a sample from which arginine was removed with
Amberlite (page 157).
.
Procedure-As the acetate with p-dimethylaminob enzaldehyde.
The
acetate must first be formed. Evaporate a volume of sample to contain
1-5 mg. of glncosamine to dryness in a porcelain evaporating dish. Add
1 ml. of 5 per cent sodium methylate, not more than 2 days old. Rub
the residue thoroughly with a glass rod and cool the dish. After 5
minutes, add 0.3 m!. of acetic anhydride dropwise. After 5 minutes
add 1 ml. of water. Transfer to a filter
and wa 'h the residue first with
1 mI. and tben witb 0.5 ml. of water. To the filtrate add 1 ml. of etbanol
and 0.5 mI. of 30 per cent sodium hydroxide solution. Let stand at
20° for 10 minutes. Heat at 100 ° for exactly 90 seconds, avoiding loss
of water by evaporation. Cool at 20° for 10 minutes. Add 3 m!. of
the pale-yellow, stable reagent which contains 0.8 gram of p-dimethyl-
amino benzaldehyde in 30 mI. of ethanol and 30 m!. of concentrated
hydrochloric acid. Read at 535 m", after 5 minutes correcting for both
reagent and sample blanks.
As a p1Jrrol der·ivative with p-dimethylaminobenzaldehyde.
Treat 2
ml. of sample containing 0.04-0.004 mg. of glucosamine per mI. with
5.5 ml. of a colorless reagent containing 0.9 per cent of acetylacetone
and 4.8 per cent of sodium carbonate, with 1: 1 hydrochloric acid added
to pH 9.8. Heat for 20 minutes in boiling water which comes to above
n-AOETYLGL UOOSAMINE 61 the level in the container. Minimize evaporation. 0001 and dilute to nearly
n-AOETYLGL UOOSAMINE
61
the level in the container. Minimize evaporation. 0001 and dilute to
nearly 25 ml. with absolute ethanol. Add 2.5 ml. of a reagent containing
0.8 gram of p-dimethylaminobenzaldehyde in 30 ml. of absolute ethanol
and 30 ml. of concentrated hydrochloric acid. Dilute to 25 ml. with
absolute ethanol and incubate at 30° for 24 hours. Read at 512 m", or
553 m", against a reagent blank.
As anhydrohexoses. To 0.5 ml. of unknown, add 0.5 ml. of 5 per cent
sodium nitrite solution, and 0.5 ml. of 1:2 acetic acid. Shake and after
10 minutes, which completes the amination, remove excess nitrous acid
by adding 0.5 ml. of 12.5 per cent ammonium sulfamate solution. Shake
repeatedly for 30 minutes. At the same time treat another 0.5 ml. of
sample in the same way but do not let it stand, so that it will not
be deaminated.
To 2 ml. of the solution which contains 0.005-0.1 mg. per ml. of
deaminated hexosamule, and to 2 ml. of the one not deaminated, add
2 rol. of 1: 20 hydrochloric acid and 0.2 ml. of 1 per cent solution of
indole in ethanoL Immerse in a boiling water bath for 5 minutes. An
intensive orange color and slight turbidity will appear. To remove the
turbidity, add 2 ml. of ethanol and shake. Make readings at 520 and
492 m", respectively. Subtract the value for Dm
- D 520 of the nonde-
aminated sample from the other and read against a calibration curve.
CHONDROSAMINE
The same pyrrol derivative and
anhydrohexose
are
formed
from
chondrosamine
as from
glucosamine
and
estimated in
the way
just
described.
n-AoETYLGLuOOSAMINE
In the enolic form, glucosamine acetate is 2-methyl-4-a,,B,y,S-tetra-
hyuroxy-n-butyl oxazole which reacts with p-dimethylaminobenzalde-
hyde. GO n-Acetylchondrosamine gives an identical reaction. Accuracy
to ±2 per cent is obtained when sample and standard do not differ by
more than 20 per cent. The color is sensitive to excess hydrochloric acid.
Procedure-As reagent dissolve 2 grams of p-dimethylamino-
benzaldehyde in 95 ml. of glacial acetic acid and 5 m!. of concentrated
hydrochloric acid. The final solution should be only pale yellow and
addition of 1 m!. of water to 9 ml. of the reagent must not intensify this.
66 Walter T. J. Morgan and Leslie A. Elson, Bioc116m J. 28, 988 -95 (1934) .
62 ALIPIIATI A1\UNES A D AMIDES Transfer a I-ml. sample containing 0.1-1 mg. and add
62 ALIPIIATI
A1\UNES A
D AMIDES
Transfer a I-ml. sample containing 0.1-1 mg. and add 0.1 ml. of 5 per
cent sodium carbonate 01 utlon. IIeat in boilillg
water for 5 minute
and cool. Add glacial acetic acid to about 8 ml. Add 1 ml. of reagent
and dilute to 10 ml. with glacial acetic acid. Mix and let stand for 45
minutes. The color i then at a maximum and will not fade for an hour.
Read against a reagent blank.
n-ACETYLCIIONDROSAMlNE
This gives the identical reaction just cited for n-acetylglucosamine.
ETHYL CARBAMATE, URETHANE
A tungstic acid blood filtrate, free from ethanol, can have its urethane
content hydrolyzed with sodium hydroxide to liberate ethano1. 67 On
distillation of the ethanol content it is oxidized by acid potassium
dichromate (Vol. III, pages 47, 49).
ISOPROPYL-N-PIIENYLOARBAMATE, IPC
The familiar oxidation of amines with hypochlorite in the presence
of phenol is applicable to determination of isopropyl-N-phenylcarba-
mate. os
Sample-Lettuce. Disintegrate 300 grams in a blender for 2 minutes.
Add 200 ml. of methylene dichloride and blend for 10 minutes, adding
chopped ice to keep the temperature below 35°. Centrifuge and separate
the solvent layer. Use 100 ml . more of solvent to rinse the samp le and
equipment. Filter and evaporate the solvent in a stream of air. Transfer
to a flask with ether and evaporate the ether. Add 10 ml. of a 91: 9
mixture of 85 per cent phosphoric acid and concentrated hydrochloric
acid, and 5 ml. of glacial acetic acid. Reflux for one hour to hydrolyz e.
Cool and dilute to 50 ml. with water. Add 60 ml. of 25 per cent sodium
hydroxide solution. Steam-distil through a still head, collecting 25-35
ml., and dilute to 50 ml. with water.
Procedure-Mix 10 ml. of sample solution with 2 drops of fresh
5 per cent calcium hypochlorite solution and 2 ml. of 12: 78 hydrochloric
67 Norwood K.
Schaffer, Francis N. LoBat:0n,
and B.
S.
Walker,
Proo.
80c.
Ezptl. Biol. Meel. 70, 420-2
(1949).
6S William E. Bissinger and Robert H. Fredenburg, J •
d8800.
Official
dur.
Chern.
34,
812-16
(1951).
QUATERNARY AMMONIUM COMPOUNDS 63 acid. After 5 minutes heat to boiling and add 5 mI.
QUATERNARY AMMONIUM COMPOUNDS
63
acid. After 5 minutes heat to boiling and add 5 mI. of fresh 5 per cent
phenol solution in 1: 19 ammonium hydroxide. Dilute with water to
20 ml. and after 15 minutes read against a reagent blank. Aniline can
be used to prepare standards and results in terms of aniline multiplied
by 1.93.
QUATERNARY
AMMONIUM
COMPOUNDS '
The cations of quaternary ammonium compounds form colored salts
in sodium carbonate solution with such indicators as bromothymol blue
and bromophenol blue. These are readily extracted by chlorinated
solvents or benzene for reading. 69 The indicator itself is insoluble in the
organic solvent and does not form extractable salts with primary, sec-
ondary, and tertiary aroines, or with alkaloids. For the reaction it is
only essential that one alkyl radical be at least 4 carbons or longer, or
is C o IIr;CII 2 -. These include the methobromide of procaine
and procaine
amide, Banthin e, the N-dietbylmethylethanolamine bromide esters of
1-benzoylcyclopropanecarboxylic acid, of 2,2-diphenylpenten-4-oic, of
a-phenoxycinnanic acid, and of 2- (o-chlorophenyl) -5,6-diydro-4H-
pyran-3 -carboxylic acid, the N- (2-hydroxyetbyl-N-methylpiperidinium
bromide ester of 1-benzoylcyclopropanecarboxylic acid, tbe 1-bydroxy-
2- (dietbylmethylamine) cyclobexane bromide ester of 2,2-dipheny}pen
ten-4-oic, and 4-benzoyl-4-carbetboxy-1,ldimethylpiperidinium bromide.
Modifications of the method make it applicable also to acetylcholine,
hexamethonium, d-tubocurarine, neostigmine, and tetraethylammonium.
Tertiary amines do not interfere. The lower limit of sensitivity is about
0.001 mg. per sample. Recovery of known quantities from dilute homo-
genates of liver, kidney, and heart was 100 ± 2 per cent.
By use of a carefully measured amount of indicator the colored
complex may be read directly without extraction. 7o By this technic
accuracy is to ±2 per cent in the range 100-500 ppm., ±5 per cent in
range 50-100 ppm. By precipitation of Eriochrome Azurol B with an
alkaline earth in the presence of a dispersing agent a fresh dispersion
of the lake is obtained. Addition of a cation-active agent alters that
09 M. E. Auerbach, Ind. E'~O. Ohem.,
Anal Ed. 15, 492 -3 (1943); Ib id. 16, 739
(1944); "Official and Tentative Methods of the Association of Official AgriculturnJ
Chemists," 7th Ed., pp. 464-6 Association of Official Agricultural Chemists, Wash-
ington D. C.
(1950);
Jean
Pien,
J.
M.
Desirant,
and Mme.
Rochelle,
Ann.
tals.
fraudes
44,
290-7
(1951);
Ruth
Mitchell
and Byron B.
Clark, Proo.
Soo.
Ea:ptl.
Bio!. Meel. 81, 105-9 (1952).
7e E. L. Colichmnn, Anal.
Ohe'l)l
19, 430 -1 (1947).
64 ALIPHATIC AMlNES AND AMIDES at once. T1 A quaternary ammonium salt on a solid
64 ALIPHATIC AMlNES AND AMIDES
at once. T1 A quaternary ammonium salt on a solid base such as paper
or cloth is reacted with dilute potassium iodide solution to form a
colored triiodide which is extracted with ethanol. 72
By oxidation with potassium permanganate some quaternary am-
monium compounds are converted to. a chloroform-soluble form, giving
a violet red. 78 This contains mangan ese, which is converted to permang-
anate and read in aqueous solution.
Samples-Bottled beverages containing frttit juices. Mix thoroughly
and measure out 50 ml. of sample. Filter on a Buchner funnel and
dilute the filtrate to 100 ml. with water. Extract the filter paper with
small portions of ethanol until no more color is extracted. Transfer
the alcoholic extract to a distilling flask, and add 10 mg. of bromophenol
blue, 2 ml. of 1:1 hydrochloric acid, and 100 ml. of water. Steam-distil
and collect a volume of distillate at least 100 ml. greater than the
volume of ethanol in the e>
'iract.
Cool the residu'e in the distilling
flask, wash with 40, 30, and 30-ml. portions of petroleum ether, and
proceed to develop with bromophenol blue on both the distillate and
the extract from the residue.
Beer. Place 100 ml. of decarbonated beer in a steam-distilling flask
and add 10 mg.
of bromophenol blue and 2 ml. of 1: 1 hydrochloric acid.
Steam-distil and collect about 200 ml. of distillate. Cool the residue
and wash with 100 and 50-ml. portions of petroleum ether. Develop
with bromophenol blue.
Table sirup. Dilute 20 grams of sample to 100 ml. with water. Add
5 mi. of 0.04 per cent bromophenol blue solution and 1 ml. of 1: 1
hydrochloric acid to an aliquot. Develop with bromophenol blue.
Eggs. Mix 12.5 ± 0.25 gram of a representative sample with 10 ml.
of water. Transfer to a 250-ml. volumetric flask, using 5-10 ml. more
of water. While twirling the flask, gradually add acetone, a little at a
time, mixing constantly, until the flask is filled to the mark. Stopper
and invert several times. After 10-15 minutes, filter, collecting 200 ml.
of filtrate. Transfer to a I-liter separatory funnel using 25 ml. of
acetone for the transfer . Add 250 ml. of water and 25 ml. of 1: 1
hydrochloric acid .and mix. Extract successively with 300, 250, 150, and
100 ml. of petroleum ether, shaking gently to avoid formation of emul-
71 John A Hart and Edward W. Lee, Tappi 34, 77·9 (1951).
72 O. B. Hager, E.
M.
Young,
T. L. Flanagan and H.
B. Walker, In.d.
Eng.
Ohem., .4.na! Ed. 19, BB5·B (1947).
78 E. Flotow, Pharm. ZentroIha.Ue 83, 181·5
(1942).
QUATERNARY AMMONIUM COMPOUNDS 65 sions. Evaporate the extract to 50-75 m}. Add 10 mg. of
QUATERNARY AMMONIUM COMPOUNDS
65
sions. Evaporate the extract to 50-75 m}. Add 10 mg. of bromophenol
blue solution and use the chlorinated solvent called for under the pro-
cedure to complete the transfer.
Milk. Mix 20 ml. of milk, 50 mI. of water, and 20 ml. of 1 per cent
acetic acid. Boil for 5 minutes and filter after several hours. Wash
the residue with six successive 10-ml. portions of water and concentrate
the filtrate to 1-2 m!. Add a few ml. of water and 5 ml. of 10 per cent
sodium carbonate solution. Filter after a few minutes and wash the
precipitate. Add 1 m!. of 0.04 per cent solution of bromophenol blue
in 0.004 per cent sodium hydroxide solution and develop by bromo-
phenol blue.
Serum or plasma. Precipitate the proteins by adding an equal
volum of 6 per cent metaphosphoric acid, let stand 10 minutes, and
centrifuge. To 3-4 ml. of supernatallt liquid add 1.5 grams of sodium
carbonate and 0.2 gram of dipotassium phosphate. Develop by bromo-
phenol blue.
Urine. If the amount of quaternary ammonium compound is too
small to permit dilution, modifications are introduced in accordance
with Table 5. Certain compounds in other media also require modifica-
of the method as shown in the table.
Procedure-By bromophenol blue. The reagent for color develop-
ment will have been added in preparation of the sample by some
technics. If alkali has not already been added, add 1 m!. of 20 per cent
sodium carbonate solution to 2 mI. of sample to bring to pH 9. Prepare
fresh daily a reagent olution containing 40 mg. of bromophenol blue
in 50 ml. of a 30 per cent dipotassium phosphate solution. Add 1 ml.
of this bromophenol blue preparation to the alkaline sample if indicator
is not already present. As solvent, wash ethylene dichloride with 0.2
volume of 4 per cent sodium hydroxide solution, then 0.2 volume of
1: 15 hydrochloric acid, followed by washing 3 times with water. Wash
isoamyl alcohol in the same way and add 3 per cent to the washed
ethylene dichloride. The solvent should be prepared each week.
Transfer the sample to a separatory funnel. Add an equal volume
of ethylene chloride and shake 3-4 minutes. If the aqueous layer shows
no excess of dye, add more before completing the extraction. Let
stand until clear, draw off lower layer into another separatory funnel
containing 10 ml. of 1 per cent sodium carbonate solution, and shake
for 3-4 minutes. If the intensity of color of the lower layer is suitable,
draw off the solvent, dry for a half-hour with anhydrous sodium
66 ALIPHATIC AMINES AND .AMIDES ~~i oM·~ § II:> lO 1.01.0 I.t:Jlt':I !.Oll':! ~ .~
66
ALIPHATIC AMINES AND .AMIDES
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QUATERNARY AMMONIUM COMPOUNDS 67 sulfate, and read at 610 mr. before separation. If too deep,
QUATERNARY AMMONIUM COMPOUNDS
67
sulfate, and read at 610 mr.
before separation.
If too deep, add more ethylene chloride
For estimation without extraction dilute the sample containing up
to 25 mg. of the quaternary ammonium compound with 1 ml. of 10
per
cent sodium carbonate solution
and exactly
2
ml .
of
0.040 per
cent
bromophenol blue solution. Read after exactly 5. minutes around
645 mr against a blank from which the dye was omitted. In the
calibration curve the color of the diluted dye corresponds to absence
of the quaternary compound.
By enochrome A zurol B. As reagent dissolve 0.2 gram of the dye
and
13.5 grams of potassium carbonate in water and dilute to a liter.
Mix
10 m!. of this solution with 10 m!. of 1.25 per cent magnesium
sulfate in 0.5 per cent aqueous polyvinyl alcohol to precipitate the
fresh lake. At once add 25 m!. of the sample solution. Read against
a reagent blank.
As the triiodide.
T ex til es and paper. Starch must be absent. The
method was developed for octadecyldimethylbenzylammonium chloride.
Immerse a 0.6-gram piece of fabric or paper containing quaternary
ammonium salts, in a mixture of 25 mI. of water and 1 ml. of 2 per
cent
potassium triiodide solution. Stir for about 20 minutes and rinse
with
10-ml. portions of 5 per cent potassium iodide solution until the
rinse solution is colorless. Usually 3 portions will be required. The
indefinite polyiodide has been reduced to the fairly stable triiodide.
Standing or excessive rinsing will give low results. Squeeze out as
much of the rinse solution as possible.
Extract the triiodide by immersing the sample in 20 ml. of ethanol
and working with that solvent. Filter through dry inorganic filter and
r ead at 450 mp. against 'an ethanol blank.
By oxidation with potassium perrnanganate.
Acidify 2 ml. of the
solution with sulfuric acid and add 1 ml. of chloroform. Add con-
centrated potassium permanganate solution titrametrically until the
color of excess persists in the aqueous layer. Separate the chloroform
layer quantitatively and evaporate to dryness. Add 1 ml. of con-
centrated nitric acid
to the r esidue and evaporate to dryness. Repeat
that operation. Take up the residue in 2 ml. of water and add a drop
of 2 per cent silver nitrate solution. Heat to boiling for 10 minutes
with 0.5 gram of moist potassium per. ulfate (Vol. II, page
394) to
destroy organic matter and oxidize manganese to permanganate. Dilute
to a volume which gives an appropriate intensity of color, read at
68 ALIPHATIC AMINES AND AlIHDES 525 mit, and compare with a calibration curve prepared with
68 ALIPHATIC AMINES AND AlIHDES
525 mit, and compare with a calibration curve prepared with the same
quaternary ammonium compound.
TRIMETHYLETHANOL AMMONIUM
HYDROXIDE,
CHOLINE
Choline in vegetable phosphatidcs is determined by hydrolysis and
precipitation of the liberated choline as the reineckate, the precipitate
being red is. olved and read. 74 By this technic accuracy to ±2 per cent
is obtainable over the range 0.64-15.5 mg. Precipitation must not be
from a solution containing more than 5 mg. per ml. or occlusion of the
reagent will cause unduly high results. Acetyl choline also reacts,
preferably after hydrolysis.
An indirect method is based on the chromium content of the pre-
cipitate. 76 By it the reineckate precipitate is oxidized with alkaline
hydrogen peroxide and the color developed with diphenylcarbazide.
Choline is precipitated by an aqueous solution of hexamtrodiphenyl-
amine and magnesium oxide and read in acetone. 76 The same reaction
is applicable to acetylcholine, carbamoylcholine, and succinyldicholine.
Sample-L ecithin. Acid hydrolysis. Hydrolyze a 0.2-gram sample
by refluxing for 15 hours with 20 ml. of 1: 2 hydrochloric acid . Cool
and extract fatty acids with
10, 10, and 5 ml. of ether . Neutralize the
aqueous layer by addition of 30 per cent sodium hydroxid e solution and
dilute to 25 ml. for the use of aliquots with the aqueous reagent.
Alkaline hydrolysis. Heat a 200-mg. sample in 2 ml. of absolute
methanol with 0.2 ml. of 30 per cent potas ium hydroxide solution in
a sealed tube at 100 0 for 1 hour. Cool to the same temperature at
which the previous volume was measured and take a 2-ml. aliquot of
the tube contents. Add 1: 35 sulfuric
acid until neutral to bromocresol
and evaporate to dryness at not over 50 0 • Take up the soluble matter ~
and transfer the insoluble portion with three successive 3-ml. portions
of benzene. Centrifuge and discard the benzene solution. Dry the
residue for 2 hours at not over 50 0 • Extract the residue with three
H Florence J. R. Beattie, Biochem. J. 30, 1554·9
(1936); M. H. Thornton and
F. K. Broom.e, Ind. Eng. Chem"., Anal, Ed. 14, 39·41 (1942); R. W. Engel, J. Bioi.
Chem. 144, 701 -10 (1942); C. Entcnman,
Alvin Taurog, and I. L. Chaikofi', Ibid.
ISS, 1a -18 (194~ ; Richard J. Winzler and Emily R. Meserve, Ib id. 159, 395-7 (1945);
Paul Fleury, Compt. rena. 226, 441-2 (1948) j
Paul Fleury and Hubert Guitnrd,
Ann.
pharnt. {mnc. 6, 252 ·4
(1948).
75 A. D. Marenzi and C. E. Cardini, J. Biol. Chem. 147,
363·70
(1943).
76 Gunnar Samuclsson, J. Pharm. aM Pharmacol. 5, 239·44 (1953).
CHOLINE 69 successive 3-ml. portions of hot absolute ethanol. Combine the extracts, dilute to 10
CHOLINE
69
successive 3-ml. portions of hot absolute ethanol. Combine the extracts,
dilute to 10 mI., centrifuge to separate any suspended matter, and use
an aliquot of the clear solution for determination
reagent.
by the methanolic
Tissue.
Extract the tissue with alcohol and ether and concentrate
the extract to low volume under reduced pressure in an atmosphere of
carbon dioxide. Redissolve the lipids in petroleum ether. Concentrate
an aliquot of this to low volume and precipitate phospholipids with
acetone and magnesium chloride.
Centrifuge, dissolve the precipitate in a mixture of methanol and
ether, and transfer to an apparatus for hydrolysis of phospholipids.
The apparatus consists of a 125-mI. conical flask having a side-arm of
2-ml. capacity set into the flask at an angle of 45 °, about 3 em. from
the top. Evaporate the methanol-ether solution to about 5 m!. and add
15 ml. of saturated barium hydroxide solution. Place on a steam bath
for 2 hours and shake frequently. Bring almost to dryne
and acidify
with 1.7 ml. of 1: 1 hydrochloric acid, heating and haking to complete
acidification. Add 15 ml. of petroleum ether and heat on the steam
bath with shaking. As the bubbling ceases, pour off the petroleum
ether phase, and retain the aqueous phase in the side-arm. Extract
twice more with 15 m!. of petroleum ether. This procedure extracts
fatty acids and the extract may be discarded. Dissolve the salt in the
flask by adding 5-8 drops of water and heating on the steam bath.
Transfer with two 2-m!. portions of 1: 9 hydrochloric acid for develop-
ment with diphenylcarbizide.
Plasma. 77 Boil 0.5 m!. of heparinized plasma with 8 mI. of 1:1
ethanol-acetone and filter. Use 5 ml. and 5 m!. of solvent to wash the
flask and filter. Concentrate the extract and washings to about 0.5 ro!.
and add 10 m!. of saturated barium hydroxide solution. Heat for
2 hours in boiling water, cool, add thymolphthalein indicator, and
neutralize with acetic acid to pH 8.9. Filter and wash the filter with
3 ml. and 3 ml. of water. Complete with the aqueous reineckate reagent.
Procedure--By aque01M reineckate reagent. Prepare a saturated
solution of ammonium reineckate, NH 4 [Cr(NHsh(SCN)4]IT 2 0, before
use each time by stirring a quantity of the salt in water for 15 minutes
and then filtering. Approximately 1 m!. of this solution will precipitate
about 10 mg. of choline.
77 V. Posborg Petersen, Scand. J. Olin. Lab. Invest. 2, 14· 20
(1950).
70 ALIPHATIC AMINES AND A1.HDES Add slowly and with stirrin g, 5 ml. of the
70 ALIPHATIC AMINES AND A1.HDES
Add slowly and with stirrin g, 5 ml. of the reagent and allow to tand
for 30 minutes with fr equent a'·itation. Centrifuge for 10 minutes and
discard the supernatant liquid. Wipe the mouth of the tube to remove
excess reineckate. Wash down the sides of the tube with 3 ml. of 1: 9
hydrochloric acid and 8 a itate to wash the precipitate. Avoid prolonged
agitation as it causes the precipitate to dissolve. Centrifuge again for
10 minutes. Again discard the liquid and wipe the tube. Dissolve the
reineckate in 10 ml. of acetone and centrifuge for 5 minutes. Read at
500-550 mp against acetone. For small amounts read in the ultraviolet
at 327 mp for greater sensitivity.
By methanoli c f' ein eckate r eag ent.
Transfer
a 5-ml. aliquot of the
sample in absolute ethanol. Adjust to approximately pH 4 and add a
few drops of a 2 per cent solution of Reinecke's salt in absolute
methanol. Choline is precipitated. til' for 2 minutes, refri"'erate for
an hour, and centrifuge. Decant and wash the residue wi t h 3 ml. of
methanol. Wash with 3 ml. of ether, then dry for 30 minutes at 37°.
Take up the residue in 5 ml. of acetone and r ead in the range 520-570
mp against a reagent blank.
By di phenylcarbazide.
The reagent is
a
solution of 0.2 per cent
diphenylcarbazide in ethanol.
It has a faint rose color which darkens
after a few days standing, but it can be u ed n everth eless.
Render an aqueous solution containin g 0.01 5-0.1 mg. of choline
slightly acid with 1: 9 hydrochloric acid and add an equal volume of
saturated aqueous ammonium reilleckate. Cool in ice water for at least
20 minutes to compl et e precipitation of choline. Centrifuge for 4
minutes. Chill the tube carriers beforehand to avoid solution of the
precipitate. Remove the supernatant liquid and wash the precipitate
with 0.5 ml. of ice-cold ethanol. Chill a few minutes, then repeat the
washing and
centrifu ging.
Dissolve the precipitate in 1 ml.
of acetone
and transfer
with 2-3 ml. of 60 per cent acetone . Add 2 ml. of water,
0.2 ml. of 10 per cent sodium hydroxide solution , and 0.1 ml. of 30
per cent hydrogen peroxide for each 0.05 mg. of choline in the sample.
IIeat the tube carefully in boiling water as rapid evaporation of
acetone induces spattering. In order to decompose the hydrogen
peroxide, allow to remain in the bath 20-30 minutes after all the acetone
has evaporated. During the heating a yellow color forms due to chro-
mate. Occasionally this color deepens, an indication that
ther
e is n ot
enough hydrogen peroxide present. In this case, add 0.1 ml. more of
30 per cent hydrogen peroxide and heat.
ACETYLCHOLINE 71 After oxidation of chromium is completed, cool, dilute with 3 ml. of water,
ACETYLCHOLINE
71
After oxidation of chromium is completed, cool, dilute with 3 ml.
of water, and add 2 ml. of 1:~ sulfuric acid and enough 0.25 per cent
ethanolic diphenylcarbazide solution to develop the full color intensity.
A violet-red color will form. Dilute to 25 ml. with water and read at
530 mp, ao-ainst a blank consisting of 2 ml. of 1: 9 sulfuric acid and 2
ml . of
the diphenylcarbazide solution diluted to 25 ml. with water.
By hexaniirod1·phenylamine.
As reagent mix 12 grams of hexanitro-
diphenylamine with 5 grams of marnesium oxide and 400 ml. of water.
After 20 hours, filter. If it becomes turbid before use, filter again.
To an ice-cold solution of 0.2-0.4 mg. of choline add 1 ml. of reao-ent.
Chill in ice for 30 minutes and filter. Wash with a saturated aqueous
solution of the precipitate and dry. Take up the precipitate in acetone,
dilute to 100 ml., and read at 415 mp. against acetone.
ACETYLCIIor"INE
Acetylcholine is rapidly converted in stoichiometric proportions to
hydroxami c acid by
hydroxylamine in alkali. 78 This is determinable
by ferric chloride in acid solution . Protein in the sample will be pre-
cipitated upon addition of the acid ion solution and is removed by
filtration or centrifugation. uch precipitation is hastened by addition
of trichloroacetic acid. Phospate, sulfate, fluoride, and oxalate are
iron-binding anions and inhibit the reaction. 70 If a suitable excess of
iron is present, the effect of phosphate is suppressed completely; sulfate
and borate exhibit little interference. A reaction with hexanitro-
diphenylamine described under choline above is also applicable.
Procedure-Prepare the reagent by mixing equal volumes of 16
per cent hydroxylamine hydrochloride and 14 per cent sodium hydroxide
solutions. This will keep for about 3 hours at room temperature. Mix
2 ml. of the reagent with 0.1 ml. of the sample solution. After about
1 minute adjust the pH to 1.2 ± 0.2 with 1 ml. of concentrated hydro-
chloric acid. Add 1 ml. of 10 per cent ferric chloride hexahydrate in
1:120 hydrochloric acid. Read the purple-brown color immediately at
540 mp As the blank repeat the procedure as described but reverse
the ord l' of addition of hydroxylamine, alkali, and acid.
78
Sblomo Hestrin, J. Bwl.
Che'ln.
180, 249· 61
(1949).
79
F. Lipmnnn and L. O. Tuttle, Ibid. 159, 21 (194 5).
72 ALIPHATIC AMINES AND .AMIDES CARBAMOYLCHOLINE A reaction with hexanitrodiphenylamine described under choline
72 ALIPHATIC AMINES AND .AMIDES
CARBAMOYLCHOLINE
A
reaction
with
hexanitrodiphenylamine
described
under
choline
(page 71) is applicable.
SUCCINYLDICHOLINE
A
reaction
with
hexanitrodiphenylamine
described
under
cholin
(page 71) is applicable.
,B-DIMETIIYLAMINOETHYL
BENZHYDltYL
ETHER,
BENADRYL
As a general reaction, organic bases react with methyl orange to
form colored complex salts which are soluble in appropriate organic
solvents. so The determination of ,B-dimethylaminoethyl
benzhydryl ether,
having the trade name of Benadryl, is based on this. 1 Methyl orange
enters the organic phase in direct proportion to the concentration of
the organic base with which it is combined.
.A. double extraction technic to avoid interferences consists of extract-
ing the Benadryl with heptane from alkaline solution, extracting th'e
heptane with dilute hydrochloric acid to get the organic base back into
the aqueous phase as its salt, and then re-extracting into ethylen
dichloride. Benzophenone and benzohydrol give no color by the methyl
orange reaction.
Samples-Plasma. Add 4 ml. of 0.4 per cent sodium hydroxide
solution to 3 ml. of ox alated plasma containing 0.001-0.01 mg. of
Benadryl per ml. Shake this with 25 mI. of heptane for 10 minutes
and centrifuge. Shake 20 ml. of this solution with 6 ml. of 1: 120
hydrochloric acid for 5 minutes. The Benadryl will be transferred to
the acid layer. Pipet off 5 ml. of the acid layer and add to it 1 ml. ~
of 4 per cent sodium hydroxide solution and 10 ml. of ethylene
dichloride. Shake for 5 minutes to transfer the Benadryl to the orll'anic
pbase. Remove the aqueous layer and use the ethylene dichloride layer
or an aliquot as sample.
Tissue. Use a motor-driven apparatus with a stainless steel rotor
to homogenize a I-gram sample of tissue containing about 0.02 mg. of
80 Bernard B. Brodie and Sidney Udenfriend, J. Bio!. Chem·. 158, 705·14 (1945).
81 E. Philip Gelvin, Thomas H. MeGavack and I. J. Drekter, BuZZ. New Yor'k
Med. Coil,. 9, 51· 5 (1946) j Wesley A. Dill and Anthony J. Glazko, J. Bio! Ohem.
179, 395·401
(1949).
BENADRYL 73 Benadryl. Add water during the homogenizing process so that the final homogenate is
BENADRYL
73
Benadryl. Add water during the homogenizing process so that the final
homogenate is about 5 ml. Dilution is a critical point in the procedure;
excessive dilution will cause a tendency to form emulsions with heptane
and insufficient dilution makes complete extraction difficult. Transfer
the homogenate to a bottle, add 5 ml. of 0.4 per cent sodium hydroxide
solution and 25 mI, of heptane, and shake for 15 minutes. After sep-
aration shake 20 ml. of the heptane
layer with 6
ml. of 1: 120 hydro-
chloric acid for 5 minutes. Add 1 ml. of 4 per cent sodium hydroxide
solution to 5 ml. of the acid and shake with 10 ml. of ethylene dichloride.
Centrifuge and take an aliquot of the ethylene dichloride layer for
color development.
U,'ine. Although urine contains a number of organic bases which
cause interference, an appropriate buffer reduces the effect. Prepare
the buffer by dissolving ]2.4 grams of boric acid and 14.9 grams of
potassium chloride in 800 ml. of water. Adjust the pH to 8 with the
glass electrode by addition of 4 per cent sodium hydroxide solution,
and dilute to 1 liter.
Make a lO-ml. urine sample alkaline with 4 per cent sodium
hydroxide solution and extract with 25 ml. of heptane for 10 minutes.
Centrifuge and then shake 20 ml. of the heptane solution with an equal
volume of the buffer solution. Discard this buffer and extract 20 ml.
of the pre-extracted heptane Jayer with 6 ml. of 1: 120 hydrochloric
acid. Mix 5 ml. of this acid layer with 1 ml. of 4 per cent sodium
hydroxide solution and 10 ml. of ethylene dichloride. Shake for 5
minutes to transfer the Benadryl to the organic phase. Separate the
ethylene dichloride layer as the sample solution.
Procedure-Saturate a 3.1 per cent boric acid solution with methyl
orange by shaking mechanically overnight. Filter off undissolved methyl
orange, wash the solution three times with ethylene dichloride, and
store in a bottle containing a layer of ethylene dichloride. Add 0.5 ml.
of the methyl orange reagent to 10 ml. of the ethylene dichloride
solution which contains up to 0.003 mg. of Benadryl and shake mech-
anically for 5 minutes. Remove as much as possible of the m.ethyl
orange layer and centrifuge the ethylene dichloride layer for about
10 minutes. Add 5 ml. of the ethylene dichloride layer to 0.5 ml. of a
solution made up of 2 m!. of concentrated sulfuric acid and 98 ml. of
absolute ethanol. Be sure not to transfer any methyl orange reagent
at this point. Mix thoroughly and read at 535 mp. against a reagent
hlank.
74 ALIPHATIC AMINES AND AMIDES SAOCHARIN One method of estimation of saccharin is by hydrolysis
74 ALIPHATIC AMINES AND AMIDES
SAOCHARIN
One method of estimation of saccharin is by hydrolysis to ammonia
and subsequent estimation by
Nessler's reagent. 82 Another is by fusion
with phenolsulfonic acid to form phenolsulfonphthalein, phenol red. 88
In general, in beverages containing less than 0.005 gram per 100 ml. the
results are high. The second method was developed for ice cream cones
and adapted to soft drinks. Accuracy is a little better than ±10 per
cent.
The pink-violet color developed with oxidized saccharin, copper
sulfate, and sodium nitrite is also applicable. s4 Fruit juices interfere,
but
otherwise results by this method are also accurate to about ±10
per
cent. A sample oxidized with potassium permanganate can be
estimated by ferric ion. SG Saccharin gives a violet precipitate with
naphthylamine and copper sulfate which in chloroform solution is
suitable for estimation. s6
Sample--.Alcoholic liquids. Heat 100 ml. of sample on a water bath
to remove ethanol. Usually evaporation to slightly less than half volume
will accomplish this. If the solution is sirupy, dilute with an equal
volume of water before evaporating. When removal of ethanol is
complete, dilute to 50 ml. and add 3 ml. of glacial acetic acid. Mix
and
add a slight excess of 20 per cent neutral lead acetate solution.
Mix
well, dilute to 100 mI., and mix. Filter, protecting from evapora-
tion, and use an aliquot for development as phenol red.
Fruit juices and sirups.
Dilute 50 ml.
of sample to about 75 m!.
with water and add 3 m!. of glacial acetic acid.
excess of 20 per cent neutral lead acetate solution.
Mix and add a slight
Mix well, dilute to
82Guiseppe
Testoni,
Z. N ahr. GenU88m . 18, 577·87 (1909); A. F. Lerrigo and
A. L. Williams, Analyst 52, 375· 83 (1927); John C. Krantz, Jr., J. A880C. Official
AUr. Chem. 1'7, 193·95 (1934); Ibid. 18, 372·3 (1935); Ibid. 19, 205·6 (1936);
, 'Official and Tentative Methods of Analysis. of the Association of OJ:Ireial Agricul·
tural Oh emists , " 7th ed., pp. 467 · 8, Associa tion of Official AgricultuIal Chemists,
Washington, D. O. (1950).
88 L.
J.
Cross
and J.
L . P erlm a n,
N.
Y
. S t at e Dept. Aor. and Mar7c et 8, Ann.
Report 1930, 89·9!); Wm. F. Reindollar, J. A8S0C. Official Aor. ahem. 24, 326·7
(1941); E. G. Whittle, Analyst 69, 45·7
(1944).
84 V. A. Rozanova, Obsllches t v cnno e P itani e 9, No . 1, 19· 25 (1941); P. A. Soifer,
Gigiena i
Sanit. 11, No.6, 33 ·6
(1946).
85 Jacques Lavagne, Ann. pharm. franc. 3, 20 · 9 (1945).
86 A. Gandini, FOITmo.co 8ci. e tec. (Pavia) 1, 34·8 (1946).
SACCHARIN 75 100 ml. and mix. use an aliquot for Filter, protecting the filtrate from
SACCHARIN
75
100 ml.
and mix.
use an aliquot for
Filter, protecting the filtrate from evaporation, and
development as phenol red.
Solid or semi-soUd p1·oducts. Add boiling water to a 50-gram
sample to dilute to 75 mL Let stand for 2 hours, shaking occasionally.
Add 3 ml. of glacial acetic acid and mix thoroughly. Add a slight
excess of 20 per cent neutral lead acetate solution. Dilute to 100 ml.
with cold water and mix. Let stand for 20 minutes, filter, protecting
from evaporation, and use an aliquot for development as phenol red.
Procedure-By Nessler's reagent. Add 2 ml. of concentrated hydro-
chloric acid to 50 ml. of sample and extract with two successive 50-mL
portions of ether. Filter the combined ether extracts through cotton
and wash with 5 ml. of distilled water acidified with a drop of con-
centrated hydrochloric acid. Evaporate the ether solution to dryness
on a water bath. Add 5 ml. of water and 6 ml. of concentrated hydro-
chloric acid to the residue. Evaporate to about 1 ml. Dilute to 50 ml.
with ammonia-free water. Add 2.5 ml. of Nessler's reagent (page 181)
and compare with a standard (Vol. II, pages 816-817) similarly treated
or read the transmittance at 420 mp. against a reagent blank. The color
is stable for about 30 minutes.
As phenol red. Extract a measured volume of alcohol·free sample
containing 0.005-0.03 gram of saccharin with ether. Evaporate the ether
extract to dryness. Fuse the residue from ether extraction with a
suitable amount of phenolsulfonic acid for 2 hours at 130-140°. Let cool
and dissolve in water. Make alkaline and dilute to a known volume.
Read at 480 mp. against a reagent blank.
By copper sUlfate and sodium nitrite. To 10 ml. of neutral, ethanol-
free sample, add a drop of 1: 3 sulfuric acid and extract with 3 suc-
cessive 5-ml. portions of ether. Combine the ether extracts and evaporate
until ethanol- and ether-free. Take up by heating with water, cool,
and dilute to 15 mL to contain not less than 0.02 per cent of saccharin .
Add 0.5 ml. of 0.5 per cent hydrogen peroxide. Add 0.5 ml. of a
reagent containing 0.3 per cent of copper sulfate pentahydrate in 1: 9
acetic acid, then 0.5 ml of 2 per cent sodium nitrite solution. Dilute
to 20 ml. and read after 20 minutes against a reagent blank.
By ferric. ion.
Extract a sample containing 1-10 mg. of saccharin
successively with three 5-ml. portions of ether.
Evaporate the filtered
extracts to dryness on a water bath and take up the residue in 10 ml.
of 1: 360 sulfuric acid.
IIeat in a water bath and add saturated potas-
76 AL I PHA T IC AMINES AND AMIDES sium permanganate dropwise until excess is
76 AL I PHA T IC AMINES AND AMIDES
sium permanganate dropwise until excess is pr esent for 2 minutes.
Decolorize with a drop or two of 10 per cent ethanol. Filter aHd wash
the filter to give 100 ml. of solution. Cool, e},,'1;ract with thrc 10-ml.
portions of ether, and filter the combined extracts through a dry paper.
Evaporate the ether and take up the residue with three 3-ml. portion
of boiling water. Cool and add 2 drops of 5 per cent olution of ferric
chloride hexahydrate and 2 drops of 6 per cent hydrogcn peroxide.
After 5 hours read against a reaO"ent blank.
ACRIFLAVINE
Acriflavine is a mixture of 2,8-diamino-10-methylacridinium chloride
and 2,8-diaminoacridine. Both give a purple to burgundy-red color with
nitric acid containing oxides of nitrogen or with sodium nitrite and
hydrocbloric acid. 87
P r oce dure-Add 1 ml. of 10
per cent nitric acid, freshly prepared
from commercial fuming acid, to 1 ml. of sample containing about
0.1-0.3 mg. of test substance. Stir vigorously until a maximum color is
developed. Dilute to 10 ml. and read against a reagent blank.
m-BENZAMINOSEMIOARBAZIDE,
CRYOGENIN
Cryogenin
gives
an
orange
color
with
concentrated
hydrochloric
acid which is mol'
intense if some chlodc acid is present. 88
Procedure-To
1
ml.
of sample